Methods of treating hypogonadism with transnasal testosterone bio-adhesive gel formulations in male with allergic rhinitis, and methods for preventing an allergic rhinitis event

ABSTRACT

The present invention relates to methods of treating hypogonadism in a male subject through administering intranasally to the male subject an intranasal testosterone bio-adhesive gel formulation to deliver a therapeutically effective amount of testosterone. In particular, the testosterone therapy of the invention remains effective if an allergic rhinitis event occurs in the male during the treatment or when the male subject uses a topical nasal vasoconstrictor or a topical intranasal decongestant during the hypogonadism treatment. Further, the present invention relates to a method of preventing the occurrence of an allergic rhinitis event in a male, who is undergoing a hypogonadism treatment with an intranasal testosterone bio-adhesive gel. In certain embodiments, the intranasal testosterone bio-adhesive gel formulation according to the invention comprises 4.0% and 4.5% testosterone.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/275,633, filed Feb. 14, 2019, which is a continuation of U.S. patent application Ser. No. 16/044,903, filed Jul. 25, 2018, which is a continuation of U.S. patent application Ser. No. 15/856,156 filed Dec. 28, 2017, which is a continuation of U.S. patent application Ser. No. 15/599,316, filed May 18, 2017, which is a continuation of U.S. patent application Ser. No. 15/284,479, filed Oct. 3, 2016, which is a continuation of U.S. patent application Ser. No. 15/045,208, filed Feb. 16, 2016, which is a continuation of U.S. patent application Ser. No. 14/753,552, filed Jun. 29, 2015, which is a continuation of U.S. patent application Ser. No. 14/536,130, filed Nov. 7, 2014, which is a continuation of U.S. patent application Ser. No. 14/215,882, filed Mar. 17, 2014, and claims the benefit of and priority to U.S. Provisional Patent Application No. 61/802,297, filed Mar. 15, 2013, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of treating hypogonadism in a male subject through administering intranasally to the male subject an intranasal testosterone bio-adhesive gel formulation to deliver a therapeutically effective amount of testosterone, thereby treating the hypogonadism. In particular, the present testosterone therapy remains effective for treating hypogonadism when an allergic rhinitis event occurs in the male or when the male subject uses a topical nasal vasoconstrictor or a topical intranasal decongestant during the hypogonadism treatment. Further, the present invention relates to a novel method of preventing the occurrence of an allergic rhinitis event in a male, who is undergoing a hypogonadism treatment with an intranasal testosterone bio-adhesive gel formulation of the present invention. In certain embodiments, the intranasal testosterone bio-adhesive gel formulation according to the present invention comprises 4.0% and 4.5% testosterone.

BACKGROUND

Androgens are a group of C19 steroids that cause masculinization of the genital tract and the development and maintenance of male secondary sex characteristics. They also contribute to muscle bulk, bone mass, libido, and sexual performance in men. Testosterone is the main androgen secreted by the Leydig cells of the testes, and its production increases during puberty. See. e.g., Tietz: Textbook of Clinical Chemistry and Molecular Diagnostics, 4th edition, Editors: Burtis C A, Ashwood E R, and Bruns D E (2006.). Androgen deficiency is now recognized to be a relatively common condition in the aging male. See, e.g., 2. Wang C, Swerdloff R. S.: Androgen replacement therapy. Ann Med, 29: 365-370 (1997); Matsumoto A. M.: Andropause: clinical implications of the decline in serum Testosterone levels with aging in men. J Gerontol A Med Sci, 57: M76-M99 (2002); and Haren Mt et al.: Andropause: a quality-of-life issue in older males. Med Clin North Am, 90: 1005-1023 (2006). Testosterone hormone therapy is indicated for replacement therapy and males having conditions associated with a deficiency or absence of endogenous testosterone, such as to treat male hypogonadism. This may cause sexual dysfunction, muscle loss, increase in fat, infertility, decreased beard and body hair and other conditions.

Hypogonadism is defined as testosterone deficiency. Male hypogonadism may be congenital or it may develop later in life due to, e.g., injury, trauma, surgery, infection, disease, drugs and/or aging. Generally, child-onset male hypogonadism has minimal consequences and generally remains undiagnosed until puberty is delayed. The symptoms or signs associated with child-onset male hypogonadism, if left untreated, include poor muscle and body hair development, including poor facial, pubic, chest and axillary hair growth, a high-pitched voice, excessive growth of arms and legs in relation to the trunk of the body, a small scrotum, abnormal phallic and testicular growth, and other growth problems, e.g., growth and maturation of the prostate and seminal vesicles. In adult-onset male hypogonadism, the symptoms may include a deficiency in spermatozoa production, osteoporosis, muscle loss or alterations in body musculature, fat distribution, fatigue and loss of energy, weakness, anemia, mood swings, e.g., depression and anger, a decline in cognitive skills, including memory loss and inability to concentrate, sleep disturbances, gynecomastia, a reduction in both beard and body hair, impotence, erectile dysfunction; a decrease in ejaculate volume, infertility, a decrease in sexual desire (loss of libido), and a regression of other secondary sexual characteristics.

Male hypogonadism is designated as either primary hypogonadism, which is due to a disorder of the testes, or central or secondary hypogonadism that results from a disorder in the hypothalamic-pituitary axis. In primary hypogonadism, there is a lack of testosterone production in the testes because the testes do not respond to FSH and LH. As a result, elevations in both hormones, FSH and LH, are observed in primary male hypogonadism. The most common cause of primary male hypogonadism is Klinefelter's syndrome. Other congenital causes of primary gonadism may include, e.g., Bilateral Congenital Anorchia, Leydig Cell Hypoplasia (Leydig Cell Aplasia), undescended testicles (Cryptorchidism), Noonan syndrome, Myotonic Dystrophy (MD) and defects in testosterone enzymatic synthesis. Causes of adult-onset primary hypogonadism may include aging, autoimmune disorders, surgery, chemotherapy, radiation, infection, disease, surgery, alcoholism, drug therapy and recreational drug use.

In secondary or central hypogonadism, insufficient amounts of FSH and LH are produced in the hypothalamus. Genital causes of secondary or central hypogonadism include, e.g., Kallmann syndrome, Prader-Willi syndrome (PWS), Dandy-Walker malformation, Isolated luteinizing hormone (LH) deficiency and Idiopathic hypogonadotropic hypogonadism (IHH). Causes of adult-onset secondary or central hypogonadism may include aging, disease, infections, tumors, bleeding, nutritional deficiencies, alcoholism, cirrhosis of the liver, obesity, weight loss, Cushing's syndrome, hypopituitarism, hyperprolactinemia, hemochromatosis, surgery, trauma, drug therapy, and recreational drug use.

In primary male hypogonadism, the levels observed for testosterone are below normal but are generally above normal for FSH and LH. In secondary or central male hypogonadism, the levels observed for testosterone, FSH and LH are below normal. Thus, diagnosis of primary or secondary male hypogonadism is typically confirmed by hormone levels and, on testing, blood levels of testosterone in both primary and secondary hypogonadism are characterized as low and should be replaced. Treatment generally varies with etiology, but typically includes testosterone replacement therapy. In the United States, testosterone may be administered as an intramuscular injection, a transdermal patch or a transdermal gel. In other countries, oral preparations of testosterone may be available.

In view of the fact that millions of men in the United States, as well as through out the world, suffer from hypogonadism, there is a real and immediate need for an effective and convenient medical therapy that can treat this disorder, so that the quality of life of these individuals can be improved. One therapeutic goal of one such therapy to solve this immediate need might be to restore testosterone levels in men to young adulthood levels in hopes to alleviate the symptoms generally associated with hypogonadism due possibly to testosterone deficiency.

SUMMARY OF THE INVENTION

The present invention offers effective methods for treating hypogonadism in a male with allergic rhinitis. In particular, the methods involve delivering a therapeutically effective amount of testosterone to the male through an intranasal administration of an intranasal testosterone bio-adhesive gel formulation. The current testosterone therapy remains effective if an allergic rhinitis event occurs in the male during the treatment. In addition, any topical nasal vasoconstrictor or topical intranasal decongestant used by the male during the hypogonadism treatment does not interfere with the efficacy of the testosterone therapy of the invention. Further, the present invention offers advantageous effects in a hypogonadism treatment, including, such as, preventing occurrence of an allergic rhinitis event in a male undergoing a hypogonadism treatment with an intranasal testosterone bioadhesive gel of the invention.

The term “a therapeutically effective amount” means an amount of testosterone sufficient to induce a therapeutic or prophylactic effect for use in testosterone replacement or supplemental therapy to treat male testosterone deficiency, namely, hypogonadism in males.

Thus, generally speaking, the present invention provides a novel method for treating hypogonadism in a male by administering intranasally to the male an intranasal testosterone bioadhesive gel formulation to deliver a therapeutically effective amount of testosterone. The hypogonadism treatment remains effective when an allergic rhinitis event occurs in the male during the treatment.

In another aspect, the invention provides a novel method of treating hypogonadism in a male, who is using a topical nasal vasoconstrictor or a topical intranasal decongestant during the treatment. In particular, the method comprises administering intranasally to the male an intranasal testosterone bio-adhesive gel formulation to deliver a therapeutically effective amount of testosterone.

The present invention also provides a novel method of preventing an allergic rhinitis event in a male, especially when the male is undergoing a hypogonadism treatment. The method of the invention comprises administering intranasally an intranasal testosterone bioadhesive gel formulation to the male to deliver a therapeutic effective amount of testosterone for treating hypogonadism.

The intranasal testosterone bioadhesive gel formulations used herein are formulated with testosterone in amounts of between about 4% and 8.0% by weight, and preferably between about 4.0% and about 4.5% by weight, and more preferably about 4.0%, about 4.5% and 8.0% by weight.

In accordance with the present invention, the rates of diffusion of the testosterone in the intranasal gel formulations of the present invention through a Franz cell membrane, as contemplated by the present invention, are between about 28 and 100 slope/mgT %, and preferably about 30 and 95 slope/mgT %. For those intranasal gels formulated with between about 4.0% and 4.5% testosterone, the preferred rates of diffusion of testosterone are between about 28 and 35 slope/mgT %.

The present invention is also directed to novel methods for pernasal administration of the nasal testosterone gels. Generally speaking, the novel methods involve depositing the intranasal testosterone gels topically into the nasal cavity of each nostril to deliver a therapeutically effective amount of testosterone in smaller volumes over dose life for providing constant effective testosterone brain and/or blood levels for use TRT, especially for effectively treating males in need of testosterone to treat hypogonadism.

More specifically, the present invention is directed to bioavailable intranasal testosterone gel formulations suitable for pernasal administration to for use in TRT and to treat hypogonadal subjects. In accordance with the present invention, and by way of example. The present invention contemplates:

Treatment with unit-dose devices pre-filled with 125 μL 4.0% testosterone gel to deliver about 5.0 mg of testosterone per nostril (intra-nasal) given, e.g., three times a day (total dose 30 mg/day); Treatment with unit-dose devices pre-filled with about 150 μL 4.5% gel to deliver about 6.75 mg of Testosterone per nostril (intra-nasal) given, e.g., twice daily (total dose 27.0 mg/day); and/or Treatment with unit-dose devices pre-filled with about 125 μL 4.5% gel to deliver about 5.625 mg of Testosterone per nostril (intra-nasal) given, e.g., three times a day (total dose 33.75 mg/day).

Generally speaking, the intranasal testosterone gel formulations of the present invention are formulated with about 4% and 4.5% testosterone by weight, and the testosterone is well absorbed when such gel formulations are administered pernasally to hypogonadal subjects. More specifically, testosterone is rapidly absorbed following pernasal administration with a peak concentration reached within 36 minutes to 1 hour 6 minutes (mean Tmax) following intra-nasal administration and maximal serum concentration is reached after about 1-2 hours post nasal administration. The maximum Testosterone concentration over a 24-hour interval is observed during the first administration (0-10 hours) in approximately 57% to 71% of the hypogonadal men while approximately 29% to 43% of the subjects had their maximum 24-h Testosterone concentration during subsequent administrations.

The formulations containing 4% and 4.5% testosterone by weight provide surprising properties. Importantly, the solubility of testosterone in castor oil pure is 3.6% maximum, falling to 3.36% about with 4% Labrafil. Addition of fumed silica (Aerosil, CabOsil) can increase the solubility of testosterone in castor oil up to 4.5% even with 4.0% Labrafil. This is counter intuitive for a person skilled in the art. However, without wishing to be bound by any particular theory, it is believed that this increase in solubility in the presence of silica is due, at least in part, to the fact that SiO₂ adsorbs about 10% of the testosterone.

In accordance with the novel methods of the present invention, the intranasal testosterone gels are topically deposited on the outer external walls (opposite the nasal septum) inside the naval cavity of each nostril, preferably at about the middle to about the upper section of the outer external wall (opposite the nasal septum) just under the cartilage section of the outer external wall inside the naval cavity of each nostril. Once gel deposition is complete within each nostril of the nose, the outer nose is then gently and carefully squeezed and/or rubbed by the subject, so that the deposited gel remains in contact with the mucosal membranes within the nasal cavity for sustained release of the testosterone over dose life. Typical testosterone gel dosage amounts deposited pernasal application is between about 50 to about 150 microliters per nostril, and preferably about 125 to about 150 microliters per nostril.

In carrying out the methods of the present invention, approximately between about 50 microliters and about 150 microliters of an intranasal testosterone gel of the present invention is applied to each nostril of a subject once or twice daily or three times a day, e.g., for one, two, three, four or more consecutive weeks, or for two, three, four, five or six consecutive days or more, or intermittently such as every other day or once, twice or three times weekly, or on demand once or twice during the same day, as TRT or to treat male testosterone deficiency, including male hypogonadism.

In addition, the present invention contemplates testosterone gel formulations for nasal administration that are pharmaceutically equivalent, therapeutically equivalent, bioequivalent and/or interchangeable, regardless of the method selected to demonstrate equivalents or bioequivalence, such as pharmacokinetic methodologies, microdialysis, in vitro and in vivo methods and/or clinical endpoints described herein. Thus, the present invention contemplates testosterone gel formulations for nasal administration that are bioequivalent, pharmaceutically equivalent and/or therapeutically equivalent, especially testosterone gel formulations for nasal administration that are 0.15% testosterone by weight of the gel formulation, 0.45% testosterone by weight of the gel formulation and 0.6% testosterone by weight of the gel formulation, when used in accordance with the therapy of the present invention to treat anorgasmia and/or HSDD by intranasal administration. Thus, the present invention contemplates: (a) pharmaceutically equivalent testosterone gel formulations for nasal administration which contain the same amount of testosterone in the same dosage form; (b) bioequivalent testosterone gel formulations for nasal administration which are chemically equivalent and which, when administered to the same individuals in the same dosage regimens, result in comparable bioavailabilities; (c) therapeutic equivalent testosterone gel formulations for nasal administration which, when administered to the same individuals in the same dosage regimens, provide essentially the same efficacy and/or toxicity; and (d) interchangeable testosterone gel formulations for nasal administration of the present invention which are pharmaceutically equivalent, bioequivalent and therapeutically equivalent.

While the intranasal testosterone gels of the present invention are preferred pharmaceutical preparations when practicing the novel methods of the present invention, it should be understood that the novel topical intranasal gel formulations and methods of the present invention also contemplate the pernasal administration of any suitable active ingredient, either alone or in combination with testosterone or other active ingredients, such as neurosteroids or sexual hormones (e.g., androgens and progestins, like testosterone, estradiol, estrogen, oestrone, progesterone, etc.), neurotransmitters, (e.g., acetylcholine, epinephrine, norepinephrine, dopamine, serotonin, melatonin, histamine, glutamate, gamma aminobutyric acid, aspartate, glycine, adenosine, ATP, GTP, oxytocin, vasopressin, endorphin, nitric oxide, pregnenolone, etc.), prostaglandin, benzodiazepines like diazepam, midazolam, lorazepam, etc., and PDEF inhibitors like sildenafil, tadalafil, vardenafil, etc., in any suitable pharmaceutical preparation, such as a liquid, cream, ointment, salve or gel. Examples of additional topical formulations for practice in accordance with the novel methods of the present invention include the topical pernasal formulations disclosed in, for example, U.S. Pat. Nos. 5,578,588, 5,756,071 and 5,756,071 and U.S. Patent Publication Nos. 2005/0100564, 2007/0149454 and 2009/0227550, all of which are incorporated herein by reference in their entireties.

The present invention is also concerned with a novel titration method to determine the appropriate daily treatment regimen, i.e., a BID or TID treatment regimen, to administer the intranasal gels of the present invention to treat hypogonadism or TRT. While the preferred treatment regimen in accordance with the present invention for administering the intranasal testosterone gels, such as 4.0% or 4.5% TBS-1 as described in Examples 1, 2, 3, 5, 7, 8, 9 and 10 above, to treat hypogonadism or TRT is twice-daily (BID) treatment regimen, the present invention contemplates that certain subjects may be more effectively treated with a three-times-a-day (TID) treatment regimen. Thus, the novel titration method of the present invention has been developed to determine which subject will require a BID or TID treatment regimen to more effectively treat hypogonadism or TRT when treated with the intranasal testosterone gels of the present invention.

In carrying out the novel titration method in accordance with the present invention, subjects will have 2 blood draws, preferably at 7 am and at 8:20 am on the test day. The day before the first blood draw, the subject will take at 10 pm, his evening intranasal dose of TBS-1. On test day, the subject will take at about 8 am, his morning intranasal dose of TBS-1.

The 24-hour C_(avg) of serum total testosterone will be estimated based on the sum of serum total testosterone levels collected at the 2 sampling points: the sample collected at about 9.0 hours (at 7 am, which is 1 hour before the morning 0800 h intranasal dose) and the sample collected at about 10.33 hours following the last evening's intranasal dose (20 minutes after the morning 0800 h dose+/−20 minutes). Note that, the blood draw times may be changed (+/−1 hour) but the delay between the last dose and the first blood draw is preferably 9 hours+/−20 minutes and the delay between the next dose administered at about 10 hours+/−20 minutes after the last dose and the second blood draw is preferably +/−20 minutes.

Testosterone serum concentrations are preferably measured by a validated method at a clinical laboratory and reported in ng/dL units.

The following titration criteria is preferably used:

If the sum of the serum total testosterone level values for PK samples collected at 9.0 hours and 10.33 hours is <755 ng/dL, then the estimated 24-hour C_(avg) for the male patient is <300 ng/dL If the sum of the serum total testosterone level values for PK samples collected at 9.0 hours and 10.33 hours is 755 ng/dL, then the estimated 24-hour C_(avg) for the male patient is ≥300 ng/dL.

With respect to those subjects with an estimated serum total testosterone C_(avg)<300 ng/dL, i.e., those subjects who sum of the serum total testosterone level values for PK samples collected at 9.0 hours and 10.33 hours is <755 ng/dL, their BID treatment regimen should be titrated to a TID treatment regimen of TBS-1 to achieve a 24-hour C_(avg) of ≥300 ng/dL. The decision to titrate the subject's daily dose to TID, however, will be made by the doctor based on the criteria specified above.

With respect to those subjects with an estimated serum total testosterone C_(avg)≥300 ng/dL, i.e., those subjects who sum of the serum total testosterone level values for pK samples collected at 9.0 hours and 10.33 hours is ≥755 ng/dL, their BID treatment regimen should remain unchanged at a BID treatment regimen of TBS-1 since their 24-hour C_(avg) is ≥300 ng/dL. The decision to titrate the subject's daily dose to TID or remain at BID, however, will be made by the doctor based on the criteria specified above.

It should be understood that, while it is preferred to draw blood from a subject to test the subject's serum total testosterone level values for pK samples at 9 hours and at 10.33 hours after the last evening's BID dose, the difference in the total draw time, i.e., 10.33 hours, may vary by as much as about +/−60 minutes and preferably no more than about +/−20 minutes between one another. It should also be understood that while, serum total testosterone level values for PK samples is 755 ng/dL is the preferred level to use to determine if titration to TID is necessary, the serum total testosterone level values for PK samples may vary as much as +/−50 and preferably no more than +/−25.

As an alternative, it should be understood that, while the titration method is described above with starting the titration method based upon the last evening's BID dose, the titration method could also be used by starting the titration method based upon the first morning dose. For example, under this alternative embodiment, the first blood draw would be taken at about 9 hours and the second blood draw would be taken at about 10.33 hours after the morning dose, so long as the second blood draw is taken at about 20 minutes after the last BID dose of the day.

Thus, a titration method in accordance with the present invention for optimizing a treatment regimen for treating a male diagnosed with hypogonadism with an intranasal testosterone gel comprises:

(a) administering intranasally to the male the intranasal testosterone gel twice daily for a selected number of days;

(b) extracting a first blood sample from the male at a selected time before a selected dose (first or second dose) of the twice daily treatment regimen on the first day after the selected number of days;

(c) extracting a second blood sample from the male at a selected time after administration of the selected dose of the twice daily treatment regimen on the first day after the selected number of days;

(d) measuring the testosterone serum level in the first blood sample to generate a first testosterone ng/dl measurement;

(e) measuring the testosterone serum level in the second blood sample to generate a second testosterone ng/dl measurement;

(f) adding the first testosterone measurement and the second testosterone measurement together to generate a serum testosterone ng/dl concentration sum for predicting a testosterone C_(avg) for the male; and

(g) comparing the serum testosterone concentration sum to a target serum testosterone level to determine an optimized intranasal treatment regimen for treating the male with the intranasal testosterone gel for maintaining in the male a testosterone 24 hour serum average at a level of at least about 300 ng/dl during the optimized treatment regimen; and

wherein, if the serum testosterone concentration sum is (i) less than the target serum testosterone level, titrating the twice daily intranasal treatment regimen for the male to a treatment regimen that is three times a day (TID) to treat the male for hypogonadism, or (ii) is equal to or greater than the target serum testosterone level, continuing with the twice daily intranasal treatment regimen for the male to treat the male for hypogonadism.

The present invention is also directed to packaged pharmaceuticals comprising the novel and improved testosterone gel formulations for nasal administration of the invention. For example, the present invention contemplates pre-filled, single or multi-dose applicator systems for pernasal administration to strategically and uniquely deposit the nasal testosterone gels at the preferred locations within the nasal cavity for practicing the novel methods and teachings of the present invention. Generally, speaking the applicator systems of the present invention are, e.g., airless fluid, dip-tube fluid dispensing systems, pumps, pre-filled, unit-dose syringes or any other system suitable for practicing the methods of the present invention. The applicator systems or pumps include, for example, a chamber, pre-filled with a single dose or multiple doses of an intranasal testosterone gel of the present invention, that is closed by an actuator nozzle or cap. The actuator nozzle may comprise an outlet channel and tip, wherein the actuator nozzle is shaped to conform to the interior surface of a user's nostril for (a) consistent delivery of uniform dose amounts of an intranasal testosterone gel of the present invention during pernasal application within the nasal cavity, and (b) deposition at the instructed location within each nostril of a patient as contemplated by the novel methods and teachings of the present invention. Preferably, when inserted into a nasal cavity, the pump design is configured to help ensure that the nasal tip is properly positioned within the nasal cavity so that, when the gel is dispensed, the gel is dispensed within the appropriate location within the nasal cavity. See Steps 3 and 8 in FIG. 10A. Additionally, the nozzles of to pumps are preferably designed to dispense the gels from the side in a swirl direction, i.e., the tips of the nozzles are designed to dispense in a side distribution direction, as opposed to a direct distribution direction, onto the nasal mucosa, as shown in steps 4 and 9 of FIG. 10A. It is believed that the swirl action allows for better gel adhesion and side distribution from the nozzle tip avoids the dispensed gel from splashing back onto the tip. Finally, it is preferred to design the nozzle and tip to allow for any residual gel on the nozzle/tip to be wiped off as the tip is removed from the nasal cavity. See, e.g., FIGS. 10A and 10 B. Examples of pre-filled, multi-dose applicator systems include, e.g., (a) the COMOD system available from Ursatec, Verpackung-GmbH, Schillerstr. 4, 66606 St. Wendel, Germany, (b) the Albion or Digital airless applicator systems available from Airlessystems, RD 149 27380 Charleval, France or 250 North Route 303 Congers, N.Y. 10950, (c) the nasal applicators from Neopac, The Tube, Hoffmann Neopac AG, Burgdorfstrasse 22, Postfach, 3672 Oberdiessbach, Switzerland, or (d) the syringes described in the Examples herein below.

A nasal multi-dose dispenser device according to embodiments of the present invention, such as the Albion or Digital airless applicator systems available from Airlessystems, is comprised of a fluid container and a distributor pump for delivery of multiple doses of a gel or other topical formulation. In one embodiment of the present invention, the nasal multi-dose dispenser device is adapted for an airless fluid dispensing system. In another embodiment of the present invention, the nasal multi-dose dispenser device is adapted for a dip tube fluid dispensing system.

An example of an airless system that is contemplated by the present invention is one that will deliver a liquid, including gel, without the need for a pressured gas or air pump to be in contact with the liquid (or gel). In general, an airless system of the present invention comprises a flexible pouch containing the liquid, a solid cylindrical container a moving piston, an aspirating pump, a dosing valve and a delivery nozzle, as depicted, for example, in FIGS. 1-4. See also FIGS. 7A, 7B, 8A, 8B, 9A, 9B, 10A, 10B and 11.

In accordance with the present invention, the multi-dose dispenser 100 of FIG. 1 is provided with a fluid container 120, a distributor pump 140 and a cap 102.

The fluid container 120 comprises a container body 122, a base 124 and a neck 126. The distributor pump 140 is fastened to the neck by a sleeve 128. The top end of the container body 122 is closed by the distributor pump 140. The sleeve 128 tightly pinches a neck gasket 150 against the top end of the container body 122. The container body 122 forms a vacuum and houses the fluid to be dispensed.

The distributor pump 140 is closed by its actuator nozzle 130, which retains the stem 144 at the stem head. The actuator nozzle 130 comprises an outlet channel 132 and tip 134.

The actuator nozzle 130 is shaped to conform with the interior surface of a user's nostril. The actuator nozzle 130 is moveable between a downward open position and upward closed position. The user removes the cap 102 and inserts the actuator nozzle 130 in the user's nostril. When the user pushes the actuator nozzle 130 downwards to the open position, fluid in the dosing chamber 180 is withdrawn by the distributor pump 140 and exits at the tip 134 via the outlet channel 132 of the actuator nozzle 130.

FIG. 2 shows a cross-sectional view of the distributor pump 140.

The distributor pump has a body 142 provided with a bottom intake having an inlet valve 160 with a ball 162 as its valve member. The ball 162 is held in place by a cage 164 and by a return spring 170.

At its bottom end, the stem 144 carries a spring cap 172. A piston 174 is located above the spring cap 172. The stem 144 passes through an axial orifice of the piston base 176.

The side walls of the piston 174 seals against the distributor pump body 142 via lips. The sleeve 128 tightly pinches a stem gasket 152 against the stem collar 146, distributor pump body 142 and top of the piston 174.

A precompression spring 178 placed between the piston base 176 and the stem collar 146. The precompression spring 178 biases the actuator nozzle 130 via the stem 144 to the closed position.

The return spring 170, which returns the piston 174 back upwards, is compressed between two opposed seats on the cage 164 and the spring cap 172.

The distributor pump 140 has a dosing chamber 180 formed between the cage 164 and piston 174. When the user pushes the actuator nozzle downwards to the open position, fluid in the dosing chamber is withdrawn by the distributor pump 140 and dispensed from the tip of the actuator nozzle 130.

When the user releases the actuator nozzle 130 upwards to the closed position, a fluid in the container body 122 is withdrawn into the dosing chamber 180 by the distributor pump 140. Thus, a dose of fluid is ready for the next actuation of the actuator nozzle by the user.

In another embodiment of the present invention, the dispenser 200 of FIG. 3 is provided with a fluid container 220, a distributor pump 240 and a cap 202.

The fluid container 220 comprises a container body 222, a base 224 and a neck 226. The distributor pump 240 is fastened to the neck by a sleeve 228. The top end of the container body 222 is closed by the distributor pump 240. The sleeve 228 tightly pinches a neck gasket 250 against the top end of the container body 222. The container body 222 houses the fluid to be dispensed.

The distributor pump 240 is closed by its actuator nozzle 230, which retains the stem 244 at the stem head. The actuator nozzle 230 comprises an outlet channel 232 and tip 234. The actuator nozzle 230 is shaped to conform with the interior surface of a user's nostril. The actuator nozzle 230 is moveable between a downward open position and upward closed position. The user removes the cap 202 and inserts the actuator nozzle 230 in the user's nostril. When the user pushes the actuator nozzle 230 downwards to the open position, fluid in the dosing chamber 280 is withdrawn by the distributor pump 240 and exits at the tip 234 via the outlet channel 232 of the actuator nozzle 230.

FIG. 4 shows a cross-sectional view of the distributor pump 240.

The distributor pump has a body 242 provided with a bottom intake having an inlet valve 260 with a ball 262 as its valve member. The ball 262 is held in place by a cage 264 and by a return spring 270. Optionally, a dip tube 290 can extend downward from the inlet valve 260 and is immersed in the liquid contained in the container body.

At its bottom end, the stem 244 carries a spring cap 272. A piston 274 is located above the spring cap 272. The stem 244 passes through an axial orifice of the piston base 276.

The side walls of the piston 274 seals against the distributor pump body 242 via lips. The sleeve 228 tightly pinches a stem gasket 252 against the stem collar 246, distributor pump body 242 and top of the piston 274.

A precompression spring 278 placed between the piston base 276 and the stem collar 246. The precompression spring 278 biases the actuator nozzle 230 via the stem 244 to the closed position.

The return spring 270, which returns the piston 274 back upwards, is compressed between two opposed seats on the cage 264 and the spring cap 272. The distributor pump 240 has a dosing chamber 280 formed between the cage 264 and piston 274. When the user pushes the actuator nozzle downwards to the open position, air enters the dosing chamber 280, which forces the fluid in the dosing chamber to be withdrawn by the distributor pump 240 and dispensed from the tip of the actuator nozzle 230.

When the user releases the actuator nozzle 230 upwards to the closed position, the air contained in the dosing chamber 280 forces the fluid in the container body 222 to be withdrawn into the dosing chamber 280. Thus, a dose of fluid is ready for the next actuation of the actuator nozzle by the user.

The amount of fluid withdrawn by the distributor pump into the dosing chamber may be a fixed volume. The distributor pumps may be of a variety of sizes to accommodate a range of delivery volumes. For example, a distributor pump may have a delivery volume of 140 μl.

The dispensers of the present invention may dispense topical intranasal gel or other topical intranasal formulations, preferably pernasally, which contain alternative or additional active ingredients, such as neurosteroids or sexual hormones (e.g., androgens and progestins, like testosterone, estradiol, estrogen, oestrone, progesterone, etc.), neurotransmitters, (e.g., acetylcholine, epinephrine, norepinephrine, dopamine, serotonin, melatonin, histamine, glutamate, gamma aminobutyric acid, aspartate, glycine, adenosine, ATP, GTP, oxytocin, vasopressin, endorphin, nitric oxide, pregnenolone, etc.), prostaglandin, benzodiazepines like diazepam, midazolam, lorazepam, etc., and PDEF inhibitors like sildenafil, tadalafil, vardenafil, etc., in the form of a liquid, cream, ointment, salve or gel. The dispensers may be suitable for cosmetic, dermatological or pharmaceutical applications. Examples of topical intranasal formulations for topical pernasal application, which can be dispensed in accordance with the present invention include the pernasal testosterone gels of the present invention or other intranasal topical gels wherein the testosterone is replaced or combined with a another active ingredient in effective amounts, such as those active ingredients discussed herein above. In addition, other testosterone formulations suitable and contemplated for dispensing from the dispensers and/or in accordance with the methods of the present invention include the formulations disclosed in, for example, U.S. Pat. Nos. 5,578,588, 5,756,071 and 5,756,071 and U.S. Patent Publication Nos. 2005/0100564, 2007/0149454 and 2009/0227550, all of which are incorporated herein by reference in their entireties.

It should be understood by those versed in this art that the amount of testosterone in a lower dosage strength intranasal testosterone gel of the present invention that will be therapeutically effective in a specific situation will depend upon such things as the dosing regimen, the application site, the particular gel formulation, dose longevity and the condition being treated. As such, it is generally not practical to identify specific administration amounts herein; however, it is believed that those skilled in the art will be able to determine appropriate therapeutically effective amounts based on the guidance provided herein, information available in the art pertaining to testosterone replacement therapy, and routine testing.

It should be further understood that the above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description further exemplifies illustrative embodiments. In several places throughout the specification, guidance is provided through examples, which examples can be used in various combinations. In each instance, the examples serve only as representative groups and should not be interpreted as exclusive examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, advantages and features of the present invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying figures and examples, which illustrate embodiments, wherein:

FIG. 1 is a side view of a first embodiment of the invention;

FIG. 2 is a cross-sectional side view of the distributor pump of the first embodiment of the invention;

FIG. 3 is a side view of a second embodiment of the invention;

FIG. 4 is a cross-sectional side view of the distributor pump of the second embodiment of the invention;

FIG. 5 is a side view of a second embodiment of the invention concerning an airless bottle assembly of the invention;

FIG. 6 is a side view of a second embodiment of the invention concerning digital actuator and rounded cap;

FIG. 7A depicts the right nostril of subject #1 after a single dose syringe administration;

FIG. 7B depicts the left nostril of subject #1 after a multiple dose dispenser administration;

FIG. 8A depicts the right nostril of subject #2 after a single dose syringe administration;

FIG. 8B depicts the left nostril of subject #2 after a multiple dose dispenser administration;

FIG. 9A depicts the right nostril of subject #3 after a single dose syringe administration;

FIG. 9B depicts the left nostril of subject #3 after a multiple dose dispenser administration;

FIGS. 10A and 10B illustrate use of a multiple dose dispenser in accordance with the present invention;

FIG. 11 illustrates a multiple dose dispenser in accordance with the present invention;

FIG. 12 depicts a Franz Cell apparatus position layouts for comparing testing in accordance with Example 5;

FIG. 13 is a graph showing the change in testosterone levels in serum over time for a 4.5% testosterone bio-adhesive gel administered in each nostril of a hypogonadal male twice daily in accordance with the present invention as compared to normal testosterone pharmacokinetics in young healthy adult males, as reported in Diver M J. et al: Diurnal rhythms of total, free and bioavailable testosterone and of SHBG in middle-aged men compared with those in young men. Clinical Endocrinology, 58: 710-717 (2003);

FIG. 14 depicts a comparison between TBS 1 A 8% (Part I);

FIG. 15 depicts a comparison between TBS 1 A 8% (Part I);

FIG. 16 depicts a comparison between 6 hours and 24 hours run (RD11101 and RD11102)

FIG. 17 depicts a comparison between TBS 1 A 4% (Part I);

FIG. 18 depicts a comparison between TBS 1 A 4% (Part II);

FIG. 19 depicts a comparison between TBS 1 A 4% (Part III);

FIG. 20 depicts a comparison slower diffusion;

FIG. 21 depicts a comparison between 6 hours and 24 hours run (RD11063 and RD11085);

FIG. 22 depicts a comparison between 400 mg and 1 gram of gel (RD11063);

FIG. 23 depicts individual amount of testosterone released from the compositions in accordance with Example 12;

FIG. 24 depicts individual testosterone concentration versus time (linear y-axis), that are grouped by subject in accordance with Example 13. Number. Black: baseline; blue: syringe; salmon: multiple dose dispenser. T=0 is at 21:00 clock-time (±30 minutes), t=12 is at 9:00 (±30 minutes) clock-time;

FIG. 25 depicts individual (blue) and median (black) testosterone concentration versus time (linear y-axis), that are grouped by treatment;

FIG. 26 depicts the probability density of the log ratio of testosterone levels that are reached with the multiple dose dispenser over levels that are reached with the syringe;

FIG. 27 depicts solubility of testosterone in different vehicles at 32° C. and at 50° C.;

FIG. 28 depicts Ternary solvent mixture optimization: Contour plot shows that, in order to achieve more than 6% testosterone solubility, higher levels of DMI and Transcutol are required;

FIG. 29 depicts a flow diagram for manufacturing TBS-1.

FIG. 30A and FIG. 30B depict a flow diagram of a manufacturing process of an intranasal testosterone gel of the present invention;

FIG. 31 depicts a mean concentration-time curves of testosterone (solid squares) and DHT (open squares) after single-dose administration of 3 different TBS-1 strengths (7.6 mg=squares; 15.2 mg=circles; 22.8 mg triangles). The lower limit of normal range for testosterone is indicated with the dashed line (based on morning serum samples);

FIG. 32 depicts testosterone diffusion rate of intranasal testosterone gel formulations of Example 13 using Franz cells method;

FIG. 33 depicts the pharmacokinetic profiles of 15 male subjects using the formulas of Example 13;

FIG. 34 is an operational diagram for manufacturing the testosterone gel formulations in accordance with the invention;

FIG. 35 depicts the mean testosterone serum concentration time profile;

FIG. 36 depicts the mean dihydrotestosterone serum concentration time profile;

FIG. 37 depicts the mean estradiol serum concentration time profile;

FIG. 38 depicts the peak response as a function of dosage;

FIG. 39 depicts the active release amount over time;

FIG. 40 depicts titration model results with testosterone Sample A taken 1 hour before morning dose and testosterone Sample B taken 20 minutes after morning dose;

FIG. 41 depicts titration model results with testosterone Sample A taken 1 hour before morning dose and testosterone Sample B taken 40 minutes after morning dose;

FIG. 42 depicts titration model results with testosterone Sample A taken 1 hour before morning dose and testosterone Sample B taken 60 minutes after morning dose;

FIG. 43 depicts titration model results with testosterone Sample A taken 1 hour before morning dose and testosterone Sample B taken 90 minutes after morning dose;

FIG. 44 depicts a linear-scale mean serum concentration time plot for testosterone;

FIG. 45 depicts a linear-scale mean serum concentration time plot for testosterone; and

FIG. 46 depicts a scale indicating testosterone and concentration vs. time curve.

DETAILED DESCRIPTION

By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description and examples are given concerning the novel lower dosage strength intranasal testosterone gels, application devices and methods of the present invention.

As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

As used herein, “at least one” is intended to mean “one or more” of the listed elements.

Singular word forms are intended to include plural word forms and are likewise used herein interchangeably where appropriate and fall within each meaning, unless expressly stated otherwise.

Except where noted otherwise, capitalized and non-capitalized forms of all terms fall within each meaning.

Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”.

All parts, percentages, ratios, etc. herein are by weight unless indicated otherwise.

As used herein, “bioequivalence” or “bioequivalent”, refers to nasally administered testosterone gel formulations or drug products which are pharmaceutically equivalent and their bioavailabilities (rate and extent of absorption) after administration in the same molar dosage or amount are similar to such a degree that their therapeutic effects, as to safety and efficacy, are essentially the same. In other words, bioequivalence or bioequivalent means the absence of a significant difference in the rate and extent to which testosterone becomes available from such formulations at the site of testosterone action when administered at the same molar dose under similar conditions, e.g., the rate at which testosterone can leave such a formulation and the rate at which testosterone can be absorbed and/or become available at the site of action to affect TRT, including hypogonadism. In other words, there is a high degree of similarity in the bioavailabilities of two testosterone gel formulation pharmaceutical products for nasal administration (of the same galenic form) from the same molar dose, that are unlikely to produce clinically relevant differences in therapeutic effects, or adverse reactions, or both. The terms “bioequivalence”, as well as “pharmaceutical equivalence” and “therapeutic equivalence” are also used herein as defined and/or used by (a) the FDA, (b) the Code of Federal Regulations (“C.F.R.”), Title 21, (c) Health Canada, (d) European Medicines Agency (EMEA), and/or (e) the Japanese Ministry of Health and Welfare. Thus, it should be understood that the present invention contemplates testosterone gel formulations for nasal administration or drug products that may be bioequivalent to other testosterone gel formulations for nasal administration or drug products of the present invention. By way of example, a first testosterone gel formulation for nasal administration or drug product is bioequivalent to a second testosterone gel formulation for nasal administration or drug product, in accordance with the present invention, when the measurement of at least one pharmacokinetic parameter(s), such as a Cmax, Tmax, AUC, etc., of the first testosterone gel formulation for nasal administration or drug product varies by no more than about ±25%, when compared to the measurement of the same pharmacokinetic parameter for the second testosterone gel formulation for nasal administration or drug product of the present invention.

As used herein, “bioavailability” or “bioavailable”, means generally the rate and extent of absorption of testosterone into the systemic circulation and, more specifically, the rate or measurements intended to reflect the rate and extent to which testosterone becomes available at the site of action or is absorbed from a drug product and becomes available at the site of action. In other words, and by way of example, the extent and rate of testosterone absorption from a lower dosage strength gel formulation for nasal administration of the present invention as reflected by a time-concentration curve of testosterone in systemic circulation.

As used herein, the terms “pharmaceutical equivalence” or “pharmaceutically equivalent”, refer to testosterone gel formulations for nasal administration or drug products of the present invention that contain the same amount of testosterone, in the same dosage forms, but not necessarily containing the same inactive ingredients, for the same route of administration and meeting the same or comparable compendial or other applicable standards of identity, strength, quality, and purity, including potency and, where applicable, content uniformity and/or stability. Thus, it should be understood that the present invention contemplates testosterone gel formulations for nasal administration or drug products that may be pharmaceutically equivalent to other testosterone gel formulations for nasal administration or drug products used in accordance with the present invention.

As used herein, “therapeutic equivalence” or “therapeutically equivalent”, means those testosterone gel formulations for nasal administration or drug products which (a) will produce the same clinical effect and safety profile when utilizing testosterone drug product for TRT and to treat testosterone deficiency, including hypogonadism, in male subjects in accordance with the present invention and (b) are pharmaceutical equivalents, e.g., they contain testosterone in the same dosage form, they have the same route of administration; and they have the same testosterone strength. In other words, therapeutic equivalence means that a chemical equivalent of a lower dosage strength testosterone formulation of the present invention (i.e., containing the same amount of testosterone in the same dosage form when administered to the same individuals in the same dosage regimen) will provide essentially the same efficacy and toxicity.

As used herein a “testosterone gel formulation for nasal administration” means a formulation comprising testosterone in combination with a solvent, a wetting agent, and a viscosity increasing agent.

As used herein, “plasma testosterone level” means the level of testosterone in the plasma of a subject. The plasma testosterone level is determined by methods known in the art.

“Diagnosis” or “prognosis,” as used herein, refers to the use of information (e.g., biological or chemical information from biological samples, signs and symptoms, physical exam findings, psychological exam findings, etc.) to anticipate the most likely outcomes, timeframes, and/or responses to a particular treatment for a given disease, disorder, or condition, based on comparisons with a plurality of individuals sharing symptoms, signs, family histories, or other data relevant to consideration of a patient's health status, or the confirmation of a subject's affliction, e.g., testosterone deficiency, including hypogonadism.

A “subject” according to some embodiments is an individual whose signs and symptoms, physical exams findings and/or psychological exam findings are to be determined and recorded in conjunction with the individual's condition (i.e., disease or disorder status) and/or response to a candidate drug or treatment.

“Subject,” as used herein, is preferably, but not necessarily limited to, a human subject. The subject may be male or female, and is preferably female, and may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. Subject as used herein may also include an animal, particularly a mammal such as a canine, feline, bovine, caprine, equine, ovine, porcine, rodent (e.g., a rat and mouse), a lagomorph, a primate (including non-human primate), etc., that may be treated in accordance with the methods of the present invention or screened for veterinary medicine or pharmaceutical drug development purposes. A subject according to some embodiments of the present invention include a patient, human or otherwise, in need of therapeutic treatment of testosterone deficiency, including hypogonadism.

“Treatment,” as used herein, includes any drug, drug product, method, procedure, lifestyle change, or other adjustment introduced in attempt to effect a change in a particular aspect of a subject's health (i.e., directed to a particular disease, disorder, or condition).

“Drug” or “drug substance,” as used herein, refers to an active ingredient, such as a chemical entity or biological entity, or combinations of chemical entities and/or biological entities, suitable to be administered to a male subject to treat testosterone deficiency, including hypogonadism. In accordance with the present invention, the drug or drug substance is testosterone or a pharmaceutically acceptable salt or ester thereof.

The term “drug product,” as used herein, is synonymous with the terms “medicine,” “medicament,” “therapeutic intervention,” or “pharmaceutical product.” Most preferably, a drug product is approved by a government agency for use in accordance with the methods of the present invention. A drug product, in accordance with the present invention, is an intranasal gel formulated with a drug substance, i.e., testosterone.

“Disease,” “disorder,” and “condition” are commonly recognized in the art and designate the presence of signs and/or symptoms in an individual or patient that are generally recognized as abnormal and/or undesirable. Diseases or conditions may be diagnosed and categorized based on pathological changes. The disease or condition may be selected from the types of diseases listed in standard texts, such as Harrison's Principles of Internal Medicine, 1997, or Robbins Pathologic Basis of Disease, 1998.

As used herein, “diagnosing” or “identifying a patient or subject having testosterone deficiency, such as hypogonadism, refers to a process of determining if an individual is afflicted with testosterone deficiency, such as hypogonadism.

As used herein, “control subject” means a subject that has not been diagnosed with testosterone deficiency or hypogonadism and/or does not exhibit any detectable symptoms associated with these diseases. A “control subject” also means a subject that is not at risk of developing testosterone deficiency or hypogonadism, as defined herein.

The testosterone gel formulations of the invention are viscous and thixotropic, oil-based formulations containing a solution of testosterone intended for intranasal application. The non-irritating formulation is designed to adhere to the inner nose. In addition, it acts as a controlling matrix, thus allowing sustained drug delivery through the nasal mucosa.

Other pharmacologically inactive ingredients in the testosterone intranasal gel are castor oil USP, oleoyl macrogolglycerides EP and colloidal silicon dioxide NF. None of these excipients are of human or animal origin. All excipients are well-known and listed in the “Inactive Ingredient” list for Approved Drug Products issued by the FDA.

The steroid hormone testosterone is the active ingredient in the testosterone gel formulations of the invention. The manufacture of the drug substance presents no potential risk for humans; the synthesis route is well-characterized,

TABLE 1 Nomenclature Testosterone INN name Testosterone Compendial name Testosterone Chemical name 17β-Hydroxyandrost-4-en-3-one Other non-proprietary Androst-4-en-3-one, 17-hydroxy-, names (17β)-Trans-testosterone Δ4-androsten-17β-ol-3-one CAS registry number 58-22-0 Proquina code 8139

Molecular Formula

C₁₉H₂₈O₂

Relative Molecular Mass

288.4

The physical chemical properties of testosterone are listed in Table 2.

TABLE 2 General Properties of Testosterone Appearance White or slightly creamy white crystals or crystalline powder. It is odourless, and stable in air. Solubility Practically insoluble in water (0.024 g/L), freely soluble in dehydrated alcohol, chloroform and in methylene chloride, soluble in dioxane and in vegetable oils; slightly soluble in ether. Melting range 153° C. to 157° C. Specific rotation +101° to +105° (dioxane) Loss on drying Not more than 1.0% UV max 238 nm Storage Protected from light

Testosterone, for testosterone gel formulations of the invention, appears as white or slightly creamy white crystals or crystalline powder. It is freely soluble in methanol and ethanol, soluble in acetone and isopropanol and insoluble in n-heptane. It can also be considered as insoluble in water (S_(20° C.)=2.41×10⁻² g/L±0.04×10⁻² g/L); its n-Octanol/Water partition coefficient (log P_(OW) determined by HPLC) is 2.84. The solubility of testosterone in oils was determined to be 0.8% in isopropylmyristate, 0.5% in peanut oil, 0.6% in soybean oil, 0.5% in corn oil, 0.7% in cottonseed oil and up to 4% in castor oil.

Because testosterone is fully dissolved within the formulations of the present invention, physical characteristics of the drug substance do not influence the performance of the drug product, testosterone gel formulations of the invention. The manufacturability of testosterone gel formulations of the invention, however is influenced by the particle size of testosterone. When using a particle size of 50% 25 microns, 90% 50 microns the solubility of the drug substance in the matrix is especially favorable.

In accordance with the present invention, the testosterone drug can be in, for instance, crystalline, amorphous, micronized, non-micronized, powder, small particle or large particle form when formulating to intranasal testosterone gels of the present invention. An Exemplary range of testosterone particle sizes include from about 0.5 microns to about 200 microns. Preferably, the testosterone particle size is in a range of from about 5 microns to about 100 microns, and the testosterone is in crystalline or amorphous and non-micronized or micronized form. Preferably, the testosterone is in crystalline or amorphous micronized form.

The molecular structure of testosterone contains no functional groups that can be protonated or deprotonated in the physiological pH-range. Therefore testosterone is to be considered as a neutral molecule with no pKa value in the range 1-14. Because it is neutral, testosterone is compatible with excipients.

The testosterone gel formulations of the invention are viscous and thixotropic, oil-based formulations containing a solution of testosterone intended for intranasal application. The non-irritating formulation is designed to adhere to the inner nose. In addition, it acts as a controlling matrix, thus allowing sustained drug delivery through the nasal mucosa.

Other pharmacologically inactive ingredients in the testosterone intranasal gel are castor oil USP, oleoyl macrogolglycerides EP and colloidal silicon dioxide NF. None of these excipients are of human or animal origin. All excipients are well-known and listed in the “Inactive Ingredient” list for Approved Drug Products issued by the FDA.

According to the “Handbook of Pharmaceutical Additives” oleoyl polyoxylglycerides are used as hydrophilic oil for topicals, injectables and nasals. In FDA-approved medicinal products it is used as co-emulsifier in topical emulsions/lotions/creams and in vaginal emulsions/creams. In France this excipient is approved for nasal preparations such as “Rhino-Sulforgan” (Laboratoire Jolly-Jatel, France; containing 10% oleoyl polyoxylglycerides) and “Huile Gomenolee 2% (“Laboratoire Goménol, France; containing 10% oleoyl polyoxylglycerides). Hence, like for castor oil it can be deduced that oleoyl polyoxylglycerides is suitable for an application route where safety and tolerability are of highest importance (e.g. injectables and nasal or vaginal preparations).

Oleoyl macrogolglycerides are also referred to as Labrafil M 1944 CS, apricot kernel oil PEG-6 esters, Peglicol-5-oleate, mixture of glycerides and polyethylene esters. The castor oil, which is used as a solvent for testosterone gel formulations of the invention, is a fixed oil. Such oils have the advantage of being non-volatile or spreading (in contrast to essential oils or liquid paraffin), but have the disadvantage of being hydrophobic. The nasal mucosa contains 95-97% water. Without the oleoyl macrogol-glycerides, the castor oil containing the active ingredient would form a non-interactive layer on the mucous membrane. In order to achieve adequate contact between the castor oil layer and the mucous membrane, the hydrophilic oleoyl macrogol-glycerides oil is added to the formulation to form an emulsion between the castor oil and the mucosa fluid.

Oleoyl macrogolglycerides are used in semi-solids at concentrations ranging from about 3 to 20%, depending on the application. The amount of oleoyl macrogol-glycerides in testosterone gel formulations of the invention is high enough to allow for a better contact of the carrier oil with the mucous membrane and low enough to have minimal impact on the amount of testosterone that can be incorporated into the carrier oil. A favourable concentration of oleoyl microgol-glycerides in testosterone gel formulations of the invention is found to be 4% of the formulation.

According to the “Handbook of Pharmaceutical Additives” colloidal silicon dioxide is used as an oil adsorbent, thermal stabiliser and gellant. In FDA-approved medicinal products it is used in dental gels, sublingual tablets, endocervical gel, suppositories, vaginal emulsions/creams/tablets/tampons and capsules for inhalation. Furthermore, it is used as an excipient in “Testoderm with adhesives” (Alza Corporation, approved in 1996) a testosterone transdermal patch. Hence, it can be deduced that colloidal silicon dioxide is suitable for an application route where safety and tolerability are of highest importance (e.g. inhalations, endocervical, vaginal or rectal preparations).

For clinical trial supplies, testosterone intranasal gel is supplied in unit-dose syringes consisting of a syringe body made from polypropylene, a plunger moulded from polyethylene and a syringe cap made from high density polyethylene. The syringes are wrapped in aluminum foil as secondary packaging. The pre-filled unit-dose syringes used in accordance with the study in the Examples are filled as follows: (a) 4% testosterone intranasal bio-adhesive gel—148 microliters and 5.92 mgs of testosterone; (b) 4.5% testosterone intranasal bio-adhesive gel—148 microliters and 6.66 mgs of testosterone; and (c) 4.5% testosterone intranasal bio-adhesive gel—148 microliters and 7.785 mgs of testosterone.

The oil in testosterone gel formulations of the invention is thickened with colloidal silicon dioxide, which acts as a gel-forming agent. This compound is used commonly for stiffening oleogels.

The intended dosage form for testosterone gel formulations of the invention is a semi-solid, not a liquid. The formulation is thickened with colloidal silicon dioxide. It is believed that colloidal silicon dioxide contributes to the thixotropic properties of the gel, simplifying drug delivery to the nostril.

Colloidal silicon dioxide is generally an inert material which is well tolerated as an excipient in mucosal applications such as suppositories. Colloidal silicon dioxide is typically used in these preparations at concentrations ranging from about 0.5 to 10%. The concentration of colloidal silicon dioxide in testosterone gel formulations of the invention is high enough to achieve gel formation but at a level that has minimal impact on testosterone incorporation into the carrier oil.

Preferably, the intranasal testosterone gels of the present invention have in general, a viscosity in the range of between about 3,000 cps and about 27,000 cps. It should nevertheless be understood by those versed in this art that, while the above-mentioned viscosity range is believed to be a preferred viscosity range, any suitable viscosities or viscosity ranges that do not defeat the objectives of the present invention are contemplated.

A detailed description of batches of a testosterone gel formulation of the invention is shown in Table 3.

TABLE 3 Composition of a testosterone gel formulation of the invention Amount Amount (% w/w) (% w/w) Component 4.0% 0.45% Testosterone 4.0%  4.5% Castor oil  88% 87.5% Oledyl macrogol- 4.0%  4.0% glycerides Colloidal silicon dioxide 4.0%  4.0%

The testosterone gel formulations of the invention are stored at room temperature (20-25° C. or 68 to 77° F.). Temperature excursions from 15 to 30° C. or 59 to 86° F. are permissible for the testosterone gel formulations of the inventions. The stability data supports a 12-month shelf life. Unit dose syringes are chosen for the primary packaging of the clinical materials for the clinical trial described below to allow for ease of dosing, ability to generate multiple doses by varying the fill volume and consistency of dose delivered. The syringe consists of a syringe body, a plunger and a syringe cap. The syringes body is moulded from polypropylene, the plunger is moulded from polyethylene and the cap is HDPE. These syringes are designed and manufactured to deliver sterile and non-sterile solutions, liquids and gels at low volumes. For additional protection from the environment (i.e., exposure to dirt, light, humidity and oxygen), the syringes are packed in a foil-laminate overwrap pouch.

The syringes and caps are designed for use in a clinical setting and meet the requirements of the EU Medical Devices Directive 93/42/EEC of Jun. 14, 1993 and as amended. As this container closure is only intended for use in this portion of the clinical program, no additional studies will be performed on the syringe and syringe components.

For a further element of protection, two syringes are contained in secondary packaging consisting of an aluminium foil pouch. Two syringes are packaged in the aluminium foil pouch and each pouch is sealed.

The pouch consists of a flexible, 3-layered-foil-laminate of a) polyester 12 micron, b) aluminum 12 micron and c) a polyethylene 75 micron. It is manufactured by Floeter Flexibles GmbH, and supplied under the name “CLIMAPAC II 12-12-75”.

The invention provides for intranasal bio-adhesive gel formulations of testosterone to be administered intranasally, wherein the dosage of the formulation is from about 4.0% or 4.5% testosterone by weight of said gel.

The methods and treatments of the present invention are suitable for TRT in men and are especially suitable to treat testosterone deficient male subjects, such as those who are diagnosed with hypogonadism.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Having now generally described the invention, the same will be more readily understood through reference to the following Examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

The following examples are put forth for illustrative purposes only and are not intended to limit the scope of what the inventors regard as their invention.

Example 1 Description and Composition of Testosterone Gel Formulations of the Invention

The compositions of three different concentrations of the drug product to be administered in this clinical trial are provided in the tables below.

Description of Dosage Form

The testosterone gel formulations of the invention are viscous and thixotropic, oil-based formulations containing solubilized testosterone intended for intranasal application. The drug product is formulated with the compendial inactive ingredients: castor oil, oleoyl polyoxylglycerides and colloidal silicon dioxide.

Two different doses of the testosterone gel formulations of the invention are intranasally administered: 0.4% w/w and 0.45% w/w. An overage is added to each syringe to account for the gel that is retained in the syringe after dosing. This overage remains consistent at 23 μl, regardless of volume of gel in the syringe.

4.0% and 4.5% Intranasal Testosterone Compositions

TABLE 1 Components, Quantity, Quality Standards and Function—4.0% testosterone gel formulation of the invention Amount Amount per Delivered Amount Syringe per Dose Quality Component (% w/w) (mg) (mg) Function Standard Testosterone 4.0% 5.92 5.0 Active USP ingredient Castor oil 88.0%  130.24 110 Solvent USP Oleoyl 4.0% 5.92 5.0 Wetting agent Ph. Eur. macrogol- (hydrophilic glycerides oil) Colloidal 4.0% 5.92 5.0 Viscosity USP/NF silicon increasing dioxide agent

TABLE 1A Components, Quantity, Quality Standards and Function—0.6% testosterone gel formulation of the invention Amount Amount per Delivered Amount Syringe per Dose Quality Component (% w/w) (mg) (mg) Function Standard Testosterone  0.6% 0.74 0.6 Active USP ingredient Castor oil 91.4%  112.42 91.4 Solvent USP Oleoyl  4.0% 4.92 4.0 Wetting agent Ph. polyoxyl- (hydrophilic Eur/NF. glycerides oil) Colloidal  4.0% 4.92 4.0 Viscosity NF silicon increasing dioxide agent Total 100% 123 mg 100 mg

TABLE 2 Components, Quantity, Quality Standards and Function, TBS-1: 5.6 mg/125 μl/syringe (4.5% gel) Amount Amount per Delivered Amount Syringe per Quality Component (% w/w) (mg) Dose (mg) Function Standard Testosterone  4.5% 6.66 5.63 Active USP ingredient Castor oil 87.5% 129.5 109.37 Solvent USP Oleoyl  4.0% 5.92 5.0 Wetting Ph. Eur. macrogol- agent glycerides (hydrophilic oil) Colloidal  4.0% 5.92 5.0 Viscosity USP/NF silicon increasing dioxide agent

TABLE 3 Components, Quantity, Quality Standards and Function, TBS-1: 6.75 mg/150 μl/syringe (4.5% gel) Amount Amount per Delivered Amount Syringe per Quality Component (% w/w) (mg) Dose (mg) Function Standard Testosterone 4.5% 7.79 6.75 Active USP ingredient Castor oil 87.5% 151.37 131.25 Solvent USP Oleoyl 4.0% 6.92 6.0 Wetting Ph. Eur. macrogol- agent glycerides (hydrophilic oil) Colloidal 4.0% 6.92 6.0 Viscosity USP/NF silicon increasing dioxide agent

Container

Testosterone gel formulations of the invention are supplied in unit-dose polypropylene syringes. Two syringes of each dosage are packaged in a protective aluminium foil pouch.

Example 2 Intranasal Testosterone Gel Formulations

The testosterone gel formulations of the invention are formulations of testosterone in an intranasal gel proposed for assessing the pharmacokinetic of two different doses of testosterone gel formulations of the invention for testosterone gel formulations of the invention in hypogonadal men.

The active ingredient, testosterone, is sourced from Bayer Schering. Challenges for nasal delivery include:

-   -   requirements for larger particles than pulmonary administration         (i.e., only particles >10 μm are sufficiently heavy to avoid         entering the respiratory tract);     -   concentrations must be higher due to the smaller volumes that         can be administered;     -   rapid clearance of the therapeutic agent from the site of         deposition results in a shorter time available for absorption;     -   potential for local tissue irritation; and     -   limited formulation manipulation possibilities to alter drug         delivery profiles.

Testosterone is indicated for TRT in males who are testosterone deficient for any number of reasons, including hypogonadism. The currently available options for administration of testosterone are oral, buccal, injectable, implantable and transdermal (patches and gels).

An intranasal testosterone (3.2%) gel is developed for the treatment of hypogonadism in men and has been administered to hypogonadal men in several clinical trials, see e.g., Mattern, C. et al., 2008 The Aging Male 11(4):171-178 (December 2008, which is incorporated herein by reference in its entirety. In a phase II study NCT00975650, which was performed in the U.S. in testosterone deficient men and which was supplemental to the Romanian study reported in Mattern et al., Supra, the 3.2% intranasal gel as reported in Mattern et al, Supra, failed to reach testosterone plasma levels required by the FDA to support TRT efficacy in testosterone deficient men. The intranasal testosterone gels formulations of the present invention are developed at concentrations of about 4.0% and 4.5% testosterone.

Example 3 Overages Testosterone Gel Formulations of the Invention

No overage is added to the formulation. An overage is added to each syringe to account for the gel that is retained in the syringe after dosing. This overage remains consistent at 23 μl, regardless of volume of gel in the syringe. The theoretical fill and dispensed amounts for testosterone gel formulations of the invention are provided below.

Theoretical Fill Theoretical Dispensed Syringe Dosage Volume (μl) Volume (μl) 4.0% Testosterone 148 125 Gel formulation of the Invention 4.5% Testosterone 148 125 Gel formulation of the Invention 4.5% Testosterone 173 150 Gel formulation of the Invention

Example 4 Physicochemical and Biological Properties Testosterone Gel Formulations of the Invention

The testosterone bio-adhesive gel formulations of the invention has a viscosity in the range of 3,000 to 10,000 mPa×sec. The viscosity is important because it facilitates maintenance of the gel in the nasal cavity in contact with the nasal mucosa. When the viscosity is less than approximately 3,000 mPa×sec (i.e., 3,000 centipoise), the gel tends to be drawn by gravity out of the nasal cavity.

Example 5 Batch Formula Testosterone Gel Formulations of the Invention

Three different concentrations of testosterone gel formulations of the invention, 0.15%, 0.45% and 0.6%, are manufactured for the proposed clinical trial. The batch formulae for these batches are presented in Table 5 below.

TABLE 5 200 KG Batch Formulae for 4.0% and 4.5% bio-adhesive testosterone gel formulations of the invention at the 8 kg Batch Size Components 4.0% 4.5% Testosterone, USP  8 g  9 g Castor oil, USP 176 g 175 g Oleoyl polyoxylglycerides, Ph.  8 g  8 g Eur./NF Colloidal silicon dioxide, NF  8 g  8 g

Example 6

The Testosterone Gel Formulations of the Invention is manufactured according to the process shown in FIG. 34.

Mixing of the Ingredients—Bulk Gel

The Pre-Mix is prepared by mixing, with a propeller mixer, the full amount of Testosterone with portion 1 of the castor oil for 10 minutes.

Mixture I is prepared by adding the Pre-Mix to the remaining castor oil and mixing for 60 minutes. The product temperature is maintained below 50° C. for the entire mixing process.

The oleoyl polyxoylglycerides are pre-heated to 40-50° C. and mixed for 10 minutes before being added to Mixture I. This is identified as Mixture II. It is mixed for 45 minutes while maintaining product temperature below 50° C. Mixture II is then screened through a sieve to remove any un-dissolved Testosterone aggregates.

Mixture III is prepared by adding the colloidal silicon dioxide to Mixture II and mixing for 15 minutes while maintaining product temperature below 50° C. A visual check is conducted after this step, to ensure that the gel is clear.

At the completion of mixing the gel is stirred and cooled to a product temperature below 30° C. The product is then discharged into stainless steel drums and the bulk gel sample is taken for analytical testing.

Filling and Packaging—Clinical Supplies

After release of the final gel mixture by the quality control laboratory, the filling and packaging process is carried out by filling a pre-determined volume into the syringe followed by the application of the syringe cap. Two syringes are packaged into a foil pouch.

The syringes are filled using a pipette with the gel taken from a holding tank. The tip of the pipette is discarded after the syringe is filled and the syringe cap is applied. Each syringe is individually labeled.

Following the application of the label, two syringes are packaged in a pre-formed foil pouch and the pouch is sealed. Each pouch is labelled.

Example 7 Drug Product TBS-1

The drug product, TBS-1, is a viscous and thixotropic, oil-based formulation containing solubilized testosterone intended for intranasal application for the treatment of hypogonadism in men.

The drug product is formulated with the following compendial inactive ingredients: castor oil, oleoyl macrogolglycerides, and colloidal silicon dioxide.

To allow for different doses to be administered in the Phase II program, a syringe is used as the unit dose container for the clinical supplies.

The syringes intended for use in the clinical program are needleless and a twist off cap is applied to the end of the syringe. The syringe consists of the syringe barrel and the plunger. The syringe barrel is formed from polypropylene. The plunger is formed from polyethylene. The syringe cap is formed from High Density Polyethylene (HDPE).

New dose formulation of TBS-1 is manufactured for clinical study TBS-1-2010-01 (submitted to the Agency on Jul. 28, 2010 Serial Number 0019). The quantity of testosterone in these formulations is 4.0% and 4.5% along with an adjustment of the amount of castor oil. The precise formulation is listed in Tables 1, 2 and 3. TBS-1 is concentrated so that the same dose is administered intranasally in a smaller volume.

Three different concentrations of TBS-1 gel will be administered in this clinical trial 5.0 mg/125 μl/syringe (4.0% gel), 5.6 mg/125 μl/syringe (4.5% gel) and 6.75 mg/150 μl/syringe (4.5% gel). An overage is added to each syringe to account for the gel that is retained in the syringe after dosing. This overage remains consistent regardless of volume of gel in the syringe.

Composition

The compositions of the three different concentrations of the drug product to be administered in this clinical trial are provided in Tables 1, 2 and 3.

TABLE 1 Components, Quantity, Quality Standards and Function, TBS-1: 5.0 mg/125 μl/syringe (4.0% gel) Amount Amount per Delivered Amount Syringe per Quality Component (% w/w) (mg) Dose (mg) Function Standard Testosterone  4.0% 2.92 5.0 Active USP ingredient Castor oil 88.0% 130.24 110 Solvent USP Oleoyl  4.0% 5.92 5.0 Wetting Ph. Eur. macrogol- agent glycerides (hydrophilic oil) Colloidal  4.0% 5.92 5.0 Viscosity USP/NF silicon increasing dioxide agent

TABLE 2 Components, Quantity, Quality Standards and Function, TBS-1: 5.6 mg/125 μl/syringe (4.5% gel) Amount Amount per Delivered Amount Syringe per Quality Component (% w/w) (mg) Dose (mg) Function Standard Testosterone  4.5% 6.66 5.63 Active USP ingredient Castor oil 87.5% 129.5 109.37 Solvent USP Oleoyl  4.0% 5.92 5.0 Wetting Ph. Eur. macrogol- agent glycerides (hydrophilic oil) Colloidal  4.0% 5.92 5.0 Viscosity USP/NF silicon increasing dioxide agent

TABLE 3 Components, Quantity, Quality Standards and Function, TBS-1: 6.75 mg/150 μl/syringe (4.5% gel) Amount Amount per Delivered Amount Syringe per Quality Component (% w/w) (mg) Dose (mg) Function Standard Testosterone 4.5% 7.79 6.75 Active USP ingredient Castor oil 87.5% 151.37 131.25 Solvent USP Oleoyl 4.0% 6.92 6.0 Wetting Ph. Eur. macrogol- agent glycerides (hydrophilic oil) Colloidal 4.0% 6.92 6.0 Viscosity USP/NF silicon increasing dioxide agent

Container

TBS-1 gel is supplied in unit-dose polypropylene syringes. Two syringes of each dosage are packaged in a protective aluminium foil pouch.

Control of Drug Products [TBS-1, Gel]

Specification [TBS-1, Gel]

The TBS-1 bulk gel is tested to the following specifications for batch release.

TABLE 1 Specification for TBS-1 Bulk Gel Test Parameter Method/Reference Acceptance Criteria Appearance Visually Slightly yellowish gel Colour of APHA colour reference Colour ≤ 250 formulation solution Viscosity Rotational viscosimeter 3,000-10,000 mPa × sec USP <911> Density Relative density 0.97-1.01 g/cm³ USP <699> Identification HPLC USP <621> Retention time corresponds to reference sample UV USP <197U> UV spectrum corresponds to reference sample Impurities HPLC USP <621> Impurity C - Epitestosterone ≤ 0.5% Impurity I - Δ-6-testosterone ≤ 0.2% Each individual unknown impurity ≤ 0.1% Total impurities ≤ 1.0% Assay HPLC USP <621> 95-105%

Finished product TBS-1 gel packaged in unit dose syringes is tested to the following specifications for batch release.

TABLE 2 Specification for TBS-1 Gel Packaged in Unit Dose Syringes Test Parameter Method/Reference Acceptance Criteria Appearance Visually Slightly yellowish gel Identification HPLC USP <621> Retention time corresponds to reference sample UV USP <197U> UV spectrum corresponds to reference sample Impurities HPLC USP <621> Impurity C - Epitestosterone ≤ 0.5% Impurity I - Δ-6-testosterone ≤ 0.2% Each individual unknown impurity ≤ 0.1% Total impurities ≤ 1.0% Assay HPLC USP <621> 95-105% Microbial limits USP <61> and <62> TAMC <10² cfu/g  TYMC <10 cfu/g P. aeruginosa 0/g S. aureus 0/g Mass variation USP <905> Complies with USP <905> TAMC—total aerobic microbial count TYMC—total combined yeast/mould count

Batch Analyses [TBS-1, Gel]

One preliminary batch (Batch No. 100304), four pilot scale batches (Batch No. ED 187, ED 188, ED 189 and ED 014), two pilot non-GMP batches (NA 090811-1 and NA090723-1) and three commercial scale (Batch 9256, 0823 and 0743) batches of TBS-1 have been produced. Data from the new batches, 0823 and 0743 are described in Tables 4 and 5.

TABLE 3 Description of TBS-1 Batches Formulation 4.0% 4.5% Batch no. 0823 0743 Batch size 200 kg 200 kg Date of manufacture June 2010 June 2010 Manufacturing site Haupt Pharma Haupt Pharma Batch no. testosterone 89100760 89100760 (Bayer/Schering) (Bayer/Schering) Equipment Commercial Process Commercial Process Filling quantity per 148 □g 173 □g container

Batch 0743, bulk 4.5% testosterone gel, is filled into two different dosage strengths, 5.6 mg (Batch 0943) and 6.75 mg (Batch 0744), by varying the weight of the gel in the finish syringe. Batch 0823, bulk 4.0% testosterone gel, is filled as one dose strength, 5.0 mg (Batch 0942).

TABLE 4 Batch Analysis - TBS-1 Batches 0743 and 0823 Test Parameter Acceptance Criteria Batch No. 0743 Batch No. 0823 Appearance of Clear, slightly yellowish Complies Complies formulation gel Colour ≤ APHA solution 250 150 150 Viscosity 3,000-10,000 mPas/30 s 5,217 5,086 Density 0.97-1.01 g/cm³ 0.99 0.99 Identification Retention time Complies 5.0 Complies 5.0 corresponds to reference min min sample UV spectrum corresponds Complies Complies to reference sample Impurities Imputity C- 0.3 0.3 Epitestosterone ≤ 0.5% Impurity I Δ-6- <0.05 <0.05 testosterone ≤ 0.2% Single impurity ≤ 0.1 <0.05 <0.05 Total impurities ≤ 1.0 0.5 0.5 Assay 95.0-105.0% 100% 100% Microbial limits TAMC < 10² cfu/g  Complies Complies TYMC < 10 cfu/g Complies Complies P. aeruginosa not detected/g Complies Complies S. aureus not detected/g Complies Complies TAMC—total aerobic microbial count TYMC—total combined yeast/mould count

TABLE 5 Batch Analysis - TBS-1 Batches 00744, 0942 and 0943 Test Acceptance Parameter Criteria 0744 0942 0943 Batch No. 0743 0823 0743 Bulk Appearance Slightly yellowish Complies Complies Complies gel Identification Retention time Complies Complies Complies corresponds to 4.9 min 5.0 min 4.9 min reference sample UV spectrum Complies Complies Complies corresponds to reference sample Impurities Impurity C ≤    0.3%    0.3%    0.3% 0.5% Impurity I ≤ <0.05% <0.05% <0.05% 0.2% Each individual   0.05%   0.05%   0.05% unknown impurity ≤ 0.1% Total impurities ≤ 1.0%    0.3%    0.3%    0.3% Assay 95-105%     99%    100%    100% Microbial TAMC < 10² cfu/g  Complies Complies Complies limits TYMC < 10 cfu/g Complies Complies Complies P. aeruginosa 0/g Complies Complies Complies S. aureus 0/g Complies Complies Complies Mass Complies with Complies Complies Complies variation USP <905>

Stability [TBS-1, Gel]

Stability Summary and Conclusions [TBS-1, Gel]

This section has been amended to include additional data on the on-going stability studies for the initial stability batches and to provide stability data on the drug product in the syringes utilized for the Phase II clinical study. Only the updated sections and new information have been included for review.

All stability studies of TBS-1 gel have been performed by ACC GmbH Analytical Clinical Concepts, Schöntalweg 9-11, 63849 Leidersbach/Aschaffenburg, Germany. Stability studies that meet ICH requirements are on-going.

TABLE 1 Stability Studies Conducted in Support of TBS-1 Container Stability Closure Drug Product Storage Data Study Study Type System Batch No. Conditions available End ICH White LDPE ED 187C 25° C./60% RH 12 months Study unit dose ED 188 40° C./75% RH  6 months completed container; ED 189 sterile air in ICH pressure EI 014 25° C./60% RH 36 months Study cushion; plus a 42 completed aluminum month pouch analysis secondary ICH package (no ED 187B 9 hours ≥ 200 Full Study Photostability nitrogen) Wh/m² (300- exposure completed 400 nm) 22 hours 1.2 Mill. Lxh. (400-800 nm) Thermal ED 188 12 hr −20° C. 4 weeks Study Cycling cycle to completed 12 hr + 40° C. ICH Syringe with Pilot Scale (non 25° C./60% RH 6 months Study Syringe Cap GMP) 40° C./75% RH completed 4.0 mg 5.5 mg 7.0 mg ICH Stainless 9256 Ambient 6 months On-going Steel Drum temperature under Nitrogen ICH Syringe with Bulk 9256 25° C./60% RH 6 months On-going Syringe Cap 9445 - 4.0 mg 40° C./75% RH 9246 - 5.5 mg 9247 - 7.0 mg ICH Stainless 0743 25° C./60% RH Initial Ongoing Steel Drum 0823 40° C./75% RH under Nitrogen ICH Syringe with 0943 25° C./60% RH initial Ongoing Syringe Cap 40° C./75% RH

Overall, stability data provided in this section are concluded to support a 24 month “use by” period for TBS-1 stored at controlled room temperature conditions [i.e., 25° C. (77° F.); excursions 15-30° C. (59-86° F.)]. The data also show that special storage conditions for the drug product are not required. The packaging configuration is adequate to protect the drug product from light and the drug product does not degrade or change physically following exposure to temperature cycling stress.

The clinical supplies are applied a 1 year re-test period, when stored at controlled room temperature conditions [i.e., 25° C. (77° F.); excursions 15-30° C. (59-86° F.)], to reflect the duration of the trial and the data available. As additional data is available the re-test period will be extended as appropriate.

Stability Data [TBS-1, Gel]

In this section, the updated stability data tables for a commercial size bulk Batch 9256, 0743 and 0823 and finish product lots 9445, 9446, 9447, 0943 are provided.

A 6 month real time stability program is ongoing on the commercial scale bulk (Batch 9256). A 36 month real time and a 6 month accelerated stability program is ongoing on three different doses of Batch 9256 packaged in 1 ml syringes: Batch 9445 4.0 mg (3.2% gel), Batch 9446 5.5 mg (3.2% gel), Batch 9447 7.0 mg (3.2% gel).

A 6 month real time stability program is underway on the commercial scale bulk batch 0743 (4.5% gel) and 0823 (4.0% gel). A 36 month real time and a 6 month accelerated stability program is underway on Batch 0943 (bulk Batch 0743 filled in 1 ml syringes).

TABLE 2 Stability Schedule for Commerical Scale Bulk TBS-1 gel and Finished Product Filled in 1 mL Syringes Storage Conditions Completed Test Intervals (° C., % RH) Product (Outstanding Test Intervals) Ambient temperature 9256 0 m, 3 m, 6 m 25 ± 2° C., 60 ± 5% 9445 0 m, 6 m (12 m, 24 m, 36 m) 40 ± 2° C., 75 ± 5% 9445 0 m, 3 m, 6 m 25 ± 2° C., 60 ± 5% 9446 0 m, 6 m (9 m, 18 m, 30 m, 36 m) 40 ± 2° C., 75 ± 5% 9446 0 m, 3 m, 6 m 25 ± 2° C., 60 ± 5% 9447 0 m, 6 m, (12 m, 24 m, 36 m) 40 ± 2° C., 75 ± 5% 9447 0 m, 3 m, 6 m 25 ± 2° C., 60 ± 5% 0943 0 m, (3 m, 9 m, 18 m, 30 m, 36 m) 40 ± 2° C., 75 ± 5% 0943 0 m, (3 m, 6 m) Ambient temperature 0743 0 m, (3 m, 6 m) Ambient temperature 0823 0 m, (3 m, 6 m)

TABLE 3 Stability Data TBS-1 Batch 9256 (3.2% Bulk Gel) Manufactured July 2009 Stored at Ambient Temperature Test 07/2009 10/2009 01/2010 Parameter Acceptance Criteria Time 0 3 months 6 months Appearance Slightly yellow gel Complies Complies Complies Colour of Colour ≤ 250 200 200 200 formulation Viscosity 3,000-10,000 mPa × 5504 5325 5198 sec Density 0.97-1.01 g/cm³ 0.99 0.99 0.99 Iodine value FIPO 78.62 77.39 76.40 Acid value FIPO ( mg KOH/g) 1.98 2.00 2.16 Peroxide FIPO (meq O₂/kg) 3.56 3.16 2.63 value Identification a. Retention time Complies Complies Complies corresponds to RS b. UV spectrum Complies Complies Complies corresponds to RS Impurities Imp C ≤ 0.5% 0.166% 0.148% 0.189% Imp I ≤ 0.1% <0.05%  0.05% <0.05% Each individual unknown  0.064%  0.05% 0.075% imp. ≤ 0.1% Total imp. ≤ 1.0% 0.230% 0.198% 0.264% Imp. D ≤ 0.2%  <0.2%  <0.2%  0.2% Assay 95.0-105%  99.4%  98.3% 100.4% Microbial TAMC <10² cfu/g  <10 cfu/g <10 cfu/g <10 cfu/g limits TYMC <10 cfu/g <10 cfu/g <10 cfu/g <10 cfu/g S.aureus 0/g Not detected/g Not detected/g Not detected/g P. aeruginosa 0/g Not detected/g Not detected/g Not detected/g

TABLE 4 Stability Data 4.0 mg TBS-1 Batch 9445 (3.2% gel) 1 ml Syringe (25 ± 2° C., 60 ± 5% RH, horizontal) Test 6 12 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 200 formulation Dissolution ≥80% within 120 min 87.8% within 120 minutes Impurities Imp C ≤ 0.5% 0.127% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.127% Imp. D ≤ 0.2%  <0.2% Assay 95.0-105%  99.3% Microbial TAMC < 10² cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g P. aeruginosa 0/g Not detected/g

TABLE 5 Stability Data 4.0 mg TBS-1 Batch 9445 (3.2% gel) 1 ml Syringe, (40 ± 2° C., 75 ± 5% RH, horizontal) Test Acceptance 3 6 Parameter Criteria Time 0 months months Appearance Slightly yellow gel Complies Complies Colour of Colour ≤ 250 200 200 formulation Dissolution ≥80% within 87.8% within 87.3% within 120 min 120 minutes 120 minutes Impurities Imp C ≤ 0.5% 0.127% 0.128% Imp I ≤ 0.1% <0.05% <0.05% Each individual <0.05% Rel RT 0.38: unknown 0.177% imp. ≤ 0.1% Rel RT 2.93: 0.066% Total imp. ≤ 1.0% 0.127% 0.371% Imp. D ≤ 0.2%  <0.2%  <0.2% Assay 95.0-105%  99.3%  99.3% Microbial TAMC < 10² cfu/g <10 cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g Not detected/g P. aeruginosa 0/g Not detected/g Not detected/g

TABLE 6 Stability Data 5.5 mg TBS-1 Batch 9446 (3.2% gel) 1 ml Syringe, (25 ± 2° C., 60 ± 5% RH, horizontal) Test Acceptance 3 6 Parameter Criteria Time 0 months mos Appearance Slightly yellow gel Complies Complies Colour of Colour ≤ 250 200 200 formulation Dissolution ≥80% within 86.8% within 83.6% within 120 min 120 minutes 120 minutes Impurities Imp C ≤ 0.5% 0.125% 0.126% Imp I ≤ 0.1% <0.05% <0.05% Each individual <0.05% <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.125% 0.126% Imp. D ≤ 0.2%  <0.2%  <0.2% Assay 95.0-105%  99.1%  99.4% Microbial TAMC < 10² cfu/g <10 cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g Not detected/g P. aeruginosa 0/g Not detected/g Not detected/g

TABLE 7 Stability Data 5.5 mg TBS-1 Batch 9446 (3.2% gel) 1 ml Syringe, (40 ± 2° C., 75 ± 5% RH, horizontal) Test Acceptance 3 6 Parameter Criteria Time 0 months months Appearance Slightly yellow gel Complies Complies Colour of Colour ≤ 250 200 200 formulation Dissolution ≥80% within 86.8% within 86.8% within 120 min 120 minutes 120 minutes Impurities Imp C ≤ 0.5% 0.125% 0.127% Imp I ≤ 0.1% <0.05% <0.05% Each individual <0.05% Rel RT 0.38: unknown 0.102% imp. ≤ 0.1% Rel RT 3.01: 0.070 Total imp. ≤ 1.0% 0.125% 0.299% Imp. D ≤ 0.2%  <0.2%  <0.2% Assay 95.0-105%  99.1%  97.9% Microbial TAMC < 10² cfu/g <10 cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g Not detected/g P. aeruginosa 0/g Not detected/g Not detected/g

TABLE 8 Stability Data 7.0 mg TBS-1 Batch 9447 (3.2% gel) 1 ml Syringe, (25 ± 2° C., 60 ± 5% RH, horizontal) Test 6 12 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 200 formulation Dissolution ≥80% within 83.5% within 120 min 120 minutes Impurities Imp C ≤ 0.5% 0.132% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.132% Imp. D ≤ 0.2%  <0.2% Assay 95.0-105%  98.7% Microbial TAMC < 10² cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g P. aeruginosa 0/g Not detected/g

TABLE 9 Stability Data 7.0 mg TBS-1 Batch 9447 (3.2% gel) 1 ml Syringe, (40 ± 2° C., 75 ± 5% RH., horizontal) Test Acceptance 3 6 Parameter Criteria Time 0 months months Appearance Slightly yellow gel Complies Complies Colour of Colour ≤ 250 200 200 formulation Dissolution ≥80% within 83.5% within 85.4% within 120 min 120 minutes 120 minutes Impurities Imp C ≤ 0.5% 0.132% 0.132% Imp I ≤ 0.1% <0.05% <0.05% Each individual <0.05% Rel RT 0.37: unknown 0.074% imp. ≤ 0.1% Rel RT 3.13: 0.069 Total imp. ≤ 1.0% 0.132% 0.275% Imp. D ≤ 0.2%  <0.2%  <0.2% Assay 95.0-105%  98.7%  99.1% Microbial TAMC < 10² cfu/g <10 cfu/g <10 cfu/g limits TYMC < 10 cfu/g <10 cfu/g <10 cfu/g S. aureus 0/g Not detected/g Not detected/g P. aeruginosa 0/g Not detected/g Not detected/g

TABLE 10 Stability Data 5.6 mg TBS-1 Batch 0943 (4.5% gel) 1 ml Syringe, (25 ± 2° C., 60 ± 5% RH, horizontal) Test 3 6 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 Complies formulation Impurities Imp C ≤ 0.5%    0.3% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.3 Assay 95.0-105%    100% Microbial TAMC < 10² cfu/g Complies limits TYMC < 10 cfu/g Complies S. aureus 0/g Complies P. aeruginosa 0/g Complies

TABLE 11 Stability Data 5.6 mg TBS-1 Batch 0943 (4.5% gel) 1 ml Syringe, (40 ± 2° C., 75 ± 5% RH, horizontal) Test 3 6 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 Complies formulation Impurities Imp C ≤ 0.5%    0.3% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.3 Assay 95.0-105%    100% Microbial TAMC < 10² cfu/g Complies limits TYMC < 10 cfu/g Complies S. aureus 0/g Complies P. aeruginosa 0/g Complies

TABLE 12 Stability Data TBS-1 Batch 0743 (4.5% gel) Bulk Stored at Ambient Temperature Test 3 6 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 Complies formulation Impurities Imp C ≤ 0.5%    0.3% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.3 Assay 95.0-105%    100% Microbial TAMC < 10² cfu/g Complies limits TYMC < 10 cfu/g Complies S. aureus 0/g Complies P. aeruginosa 0/g Complies

TABLE 14 Stability Data TBS-1 Batch 0823 (4.5% gel) Bulk Stored at Ambient Temperature Test 3 6 Parameter Acceptance Criteria Time 0 months months Appearance Slightly yellow gel Complies Colour of Colour ≤ 250 Complies formulation Impurities Imp C ≤ 0.5%    0.3% Imp I ≤ 0.1% <0.05% Each individual <0.05% unknown imp. ≤ 0.1% Total imp. ≤ 1.0% 0.3 Assay 95.0-105%    100% Microbial TAMC < 10² cfu/g Complies limits TYMC < 10 cfu/g Complies S. aureus 0/g Complies P. aeruginosa 0/g Complies

Example 8 Phase 2 Study Designed to Investigate the Intranasal Absorption of 4% of the Drug Three Times a Day and 4.5% of the Drug Administered Twice a Day and Three Times a Day

This is a Phase 2 study designed to investigate the intranasal absorption of 4% of the drug three times a day and 4.5% of the drug administered twice a day and three times a day, and to compare the absorption from the previous study in the same subjects that responded with a 3.2% testosterone gel. In the previous study, Nasobol-01-2009, a 3.2% Testosterone gel is used to deliver 4.0 mg, 5.5 mg and 7.0 mg of Testosterone intra-nasally using gel volumes of 125 μL, 172 μL and 219 μL, respectively. In this study, 5.0 mg, 5.65 mg and 6.75 mg of Testosterone is administered in gel volumes of 125 μL, 125 μL, and 150 μL, respectively. This study allowed investigating the delivery of similar Testosterone amounts in much smaller volumes.

In this open label study, subjects are equally randomized into three treatment arms. The treatments are administered for one week, in a parallel design. At the end of one week, the three treatments are compared by conducting a 24-hour pharmacokinetic investigation of the systemic absorption of the drug product testosterone and its two physiological metabolites dihydrotestosterone and estradiol.

8. Study Objectives

8.1 Primary Objective

The primary objective of this study is to determine the bioavailability through PK analysis of a 4% TBS-1 gel (applied three times a day) and 4.5% TBS-1 gel (applied twice a day and three times a day) in hypogonadal men.

8.2 Secondary Objective

The secondary objective of the study is to establish the safety profile for TBS-1.

9. Investigational Plan

9.1 Overall Study Design and Plan Description

This is an open label, randomized, balanced, three treatment (4.0% t.i.d. 4.5% b.i.d. and 4.5% t.i.d.), parallel design, pharmacokinetic study of TBS-1, administered intra-nasally. The serum concentrations of total Testosterone, Dihydrotestosterone and Estradiol are measured using validated LC/MS methods.

Hypogonadal subjects are required to visit the Clinic on three (3) occasions, of which one (1) visit (Visit 3) required an overnight stay for the previously described 24-hour pharmacokinetic profile.

The following pharmacokinetic parameters are determined for all subjects:

-   -   AUC_(0-τ), C_(avg), C_(min), C_(max), t_(max), PTF and PTS means         and standard error of the means are calculated for the 24-hour         interval.     -   The percentage of subjects with a C_(avg) for Testosterone,         Dihydrotestosterone and Estradiol, below, within and above the         Reference Range for the respective analyte is calculated.

Erythrocytosis, anemia and infections are monitored by measuring complete blood counts at screening and the Close-Out visit.

It is planned to enroll approximately 30 subjects. Twenty-two (22) subjects completed the study. Study participation is 2 to 3 weeks.

9.2 Discussion of Study Design

Testosterone therapy for hypogonadal men should correct the clinical abnormalities of Testosterone deficiency, including disturbances of sexual function. Testosterone decreases body fat and increases lean muscle mass and bone density with minimal adverse effects.

There are several Testosterone replacement products available, which can be given intra-muscularly, orally, as a buccal tablet to the gums, or topically as a patch or gel. Current replacement therapies have certain drawbacks. Testosterone injections show wide fluctuations in serum Testosterone levels often at values above the reference range (5). Testosterone patches have a high rate of skin irritation (6,7). Testosterone gels although popular in North America are not always convenient and have a risk of skin-to-skin transfer to family members (8,9). Oral Testosterone undecanoate needs to be administered with a high fat meal and levels obtained are often low (10-12).

Intra-nasal administration of a new formulation of Testosterone (TBS-1) has been shown to be effectively absorbed and shows excellent potential as a therapeutic product in the treatment of male hypogonadism (13). The nasal mucosa offers an alternative route of administration that is not subject to the first pass effect, has high permeability and ease of administration with rapid absorption into the systemic circulation producing high plasma levels similar to those observed after intravenous administration.

The advantages of the Testosterone nasal gel, when compared to other formulations, are the following: Convenient application form permitting inconspicuous use, the much smaller amount of active ingredient needed for the subject, and knowing that this type of administration is less likely to contaminate other family members (wife and children).

Several studies have indicated the utility of testosterone administration using the nasal gel. The prior study conducted in 2009 is to demonstrate the efficacy of TBS-1 in the treatment of hypogonadal men requiring Testosterone replacement therapy. Efficacy is determined by establishing an optimal pharmacokinetic profile for serum Testosterone levels following a multiple-dose b.i.d. dosing profile for TBS-1, using three different strengths of Testosterone (8.0 mg, 11.0 mg and 14.0 mg) and comparing it to that of the active control, Androderm®. The secondary objective of this study is to establish a safety profile for TBS-1. This is to be achieved by monitoring adverse and serious adverse events during the course of the entire study, and comparing various safety parameters at follow-up to those obtained at baseline. These safety parameters consisted of vital signs, complete blood counts, a chemistry profile, an endocrine profile, and urinalysis. In addition, changes to the nasal mucosa and to the prostate at follow up are compared to baseline.

An important advantage of the power of the dose finding design of this study is that it minimizes the subject selection bias and the different host groups often observed in sequential study designs.

The three clinical sites are monitored by Schiff & Company to ensure the safety of the Subjects and performance of the clinical study according to ICH E6 and FDA guidelines.

A central laboratory is used for the analysis of hematology and biochemistry parameters in order to obtain consistent and unbiased laboratory results. A second central laboratory is used for the PK analysis.

The following are the specific activities in the study design during the subject visits:

In/Ex PERIOD Day 1 Day 7 Day 8 Visit Number: 1 2 3 PROCEDURE Informed Consent¹ x Medical History x Physical Exam* & Vital Signs x x x x Subject Demographic Data x x PROCEDURE Otorhinolaryngological Exam x x Prostate Exam² x x Chemistry Profile³ x x Hematology Profile⁴ x x Urinalysis⁵ x x Serum PSA x x Hepatitis B, C, & HIV Testing x Urine Drug Screen⁶ x Ethanol Test⁷ x Hemoglobin A₁c x Serum Testosterone⁸ x Serum T, DHT & Estradiol x Serum T, DHT & Estradiol PK x Concomitant Medications x x x x Adverse Event Recording x x x x *Physical Exam on Screen and Day 8 only. Informed consent will be signed prior to Screening Visit 1 In/Ex Period: Inclusion, and Exclusion Period ²If subject had a prior normal prostate exam in Nasobol-01-2009, it will not be required. ³Chemistry Profile: Na/K, Glucose, Urea, Creatinine, Total Bilirubin, Albumin, Calcium, Phosphate, Uric Acid, AST, ALT, ALP, GGT and CK. ⁴Complete Blood Count and Differential. ⁵Urine dipstick (no microscopic). ⁶Cocaine, Cannabinoids, Opiates, Benzodiazepines. ⁷Urine alcohol by dipstick. ⁸Serum Testosterone, Dihydrotestosterone & Estradiol will be measured by a reference lab using a validated LC-MS/MS method, for T and DHT and a validated LC-MS/MS or immunoassay method, for Estradiol.

Screening Visit 1

-   -   Subjects, after having voluntarily signed the Informed Consent         Form, are interviewed by the Clinical Investigator or his/her         designee Physician/Nurse Practitioner who took the medical and         physical history, record demographic data, and performed a         routine physical examination. Body weight and Height is measured         and BMI calculated. Vital signs (seated 5 minutes) are measured         (Blood Pressure, Heart Rate, Respiratory Rate, and Body         Temperature).     -   If the subject had a normal digital rectal exam of the prostate         in the recent Nasobol-01-2009 trial, it is not repeated.     -   The Clinical Investigator assessed the subject study eligibility         based on the inclusion/exclusion criteria, and eligible subjects         that are currently on Testosterone replacement therapy needed to         undergo a wash-out period; four (4) weeks for depot products         administered intra-muscularly (e.g., Testosterone enanthate 200         mg/mL), and two (2) weeks for products administered orally or         topically (patch, gel, or buccal). At the end of the wash-out         period, subjects are to return to have their serum Testosterone         measured.     -   Treatment naïve subjects did not require a wash-out period.     -   Blood for serum Testosterone is drawn under fasting conditions,         at 0900 h±30 minutes. The serum Testosterone level must be >150         ng/dL, and <300 ng/dL.     -   Blood is drawn for Clinical Laboratory investigations after an         overnight fast (8-10 hour fast) and included the following:         -   Complete Blood Count (Hemoglobin, Hematocrit, MCV, MCHC,             RBC, WBC & Differential)         -   Clinical Chemistry profile (Na/K, Glucose, Urea, Creatinine,             Total Bilirubin, Albumin, Calcium, Phosphate, Uric Acid,             AST, ALT, ALP, GGT and CK)         -   Serum PSA         -   Testing for HBV, HCV and HIV (Hepatitis B surface antigen,             Hepatitis C antibody, HIV antibodies)         -   Whole blood sample for Hemoglobin A1c         -   Urine for dipstick urinalysis         -   Urine for Drug screen (Cocaine, Cannabis, Opiates and             Benzodiazepines). Subjects with positive test are not             enrolled, unless the positive test is due to interference             from a drug prescribed by a Physician         -   Urine for alcohol testing     -   The otorhinolaryngologic nasal endoscopy examination is done by         an ENT specialist.     -   Subjects that met all of the inclusion and exclusion criteria         are enrolled into the study and randomized into one of three         treatment groups (A, B or C).

Visit 2 (Day 1)

-   -   Subjects arrived at the Clinic under fasting conditions (6-8         hour fast) at 2000 hours or earlier.     -   Instructions are given to subjects on the proper technique for         intra-nasal dosing of TBS-1.     -   Blood is drawn at 2045 hours for baseline serum Testosterone,         Dihydrotestosterone, and Estradiol concentrations.     -   Vital Signs (seated 5 minutes) are measured (Blood Pressure,         Heart Rate, Respiratory Rate, and Body Temperature) to establish         a baseline.     -   Subjects are given a one week supply of pouches: 18 pouches for         treatment A, 12 pouches for treatment B, and 18 pouches for         treatment C. Pouches required for dosing during the         pharmacokinetic profile remained with the Clinical Investigator.         Each pouch contained two syringes pre-filled with TBS-1 gel for         treatment A, B, or C.     -   Subjects administered their first dose of TBS-1 at 2100 hours         according to their treatment group.     -   Vital Signs are measured at 2200 hours and subjects are sent         home with their supply of pouches for their treatment group.

Telephone Check (Day 4)

On Day 4, all subjects are called to check compliance of study drug administration, compliance to abstention from alcohol for 48 hours, and to document any adverse events that may have occurred. Subjects are reminded to bring in all syringes for counting at Visit 3.

Visit 3 (Day 7)

-   -   Subjects arrived at the Clinic under fasting conditions (6-8         hour fast) at 2000 hours or earlier.     -   Blood is drawn at 2045 hours for baseline serum Testosterone,         Dihydrotestosterone, and Estradiol concentrations.     -   Subject underwent a 24-hour pharmacokinetic profile immediately         after the 2100 hour dosing. Vital signs are recorded hourly for         two hours post dosing.     -   Safety parameters are recorded.     -   Subjects remained fasting for two hours post dose and then given         supper. After supper, the subjects again fasted overnight and         remained fasting until 0900 hours on Day 8. Lunch and supper on         Day 8 occurred at the regular times and are not subject to         fasting conditions.

Pharmacokinetic Blood Draws

-   -   Administration of the drug should have occurred at ±5 minutes         from the indicated time (2100 h and 0700 h for b.i.d. dosing and         2100 h, 0700 h and 1300 h for t.i.d. dosing).     -   Blood draws should have been within ±5 minutes from the         indicated times when blood draw intervals are 30 minutes and         within ±15 minutes when blood draws are >30 minutes.     -   Treatment A: Blood draws for serum Testosterone,         Dihydrotestosterone, and Estradiol measurements: Blood draws for         t.i.d. dosing are done at the following times after the 2100         hour drug administration; 0.33, 0.66, 1.0, 1.5, 2.0, 3.0, 6.0,         9.0, 9.75, 10.33, 10.66, 11.0, 11.5, 12.0, 13.0, 14.0, 15.75,         16.33, 16.66, 17.0, 17.5, 18.0, 20.0, 22.0 and 24.0 hours,         (total blood draws; 25+baseline).     -   Treatment B: Blood draws for serum Testosterone,         Dihydrotestosterone, and Estradiol measurements: Blood draws for         b.i.d. dosing are done at the following times after the 2100         hour drug administration; 0.33, 0.66, 1.0, 1.5, 2.0, 3.0, 6.0,         9.0, 9.75, 10.33, 10.66, 11.0, 11.5, 12.0, 13.0, 16.0, 19.0,         22.0, and 24.0 hours, (total blood draws; 19+baseline).     -   Treatment C: Blood draws for serum Testosterone,         Dihydrotestosterone, and Estradiol measurements: Blood draws for         t.i.d. dosing are done at the following times after the 2100         hour drug administration; 0.33, 0.66, 1.0, 1.5, 2.0, 3.0, 6.0,         9.0, 9.75, 10.33, 10.66, 11.0, 11.5, 12.0, 13.0, 14.0, 15.75,         16.33, 16.66, 17.0, 17.5, 18.0, 20.0, 22.0 and 24.0 hours,         (total blood draws; 25+baseline).     -   The last blood draw in the pharmacokinetic profile included         enough blood to measure the clinical laboratory safety         parameters required at Close-out.

Visit 3 (Day 8), Close Out Visit Subjects Underwent the Following Assessments:

-   -   A routine physical examination including vital signs (Blood         Pressure, Heart Rate, Respiratory Rate, and Body Temperature).     -   Otorhinolaryngologic nasal examination.     -   Blood sample is taken for a Complete Blood Count (Hemoglobin,         Hematocrit, RBC, WBC and differential, MCV, MCHC).     -   Blood sample for Chemistry Profile (Na/K, glucose, urea,         creatinine, calcium, phosphate, uric acid, total bilirubin,         albumin, AST, ALT, ALP, GGT, and CK).     -   Blood sample for PSA.     -   Urine sample for dipstick urinalysis.

9.3 Selection of Study Population

Subjects are included in the study according to the following inclusion/exclusion criteria:

9.3.1 Inclusion Criteria

-   -   1. Males who are responders to high-dose intra-nasal         Testosterone in the Nasobol-01-2009 trial.     -   2. Written informed consent.     -   3. Males between 18 and 80 years of age.     -   4. Men with primary or secondary hypogonadism and a morning         (0900 h±30 minutes) serum Testosterone levels >150 ng/dL and 300         ng/dL, on blood drawn under fasting conditions.     -   5. BMI between 18.5-35 kg/m².     -   6. All clinical laboratory assessments at the Screening Visit         are from blood drawn or urine collected following an overnight         fast (10 hours), and are within ±15% of the Clinical         Laboratory's reference range, except for serum Testosterone.     -   7. Normal Otorhinolaryngological nasal endoscopy examination.         See Appendix 16.1.1 for exclusion criteria pertaining to         endoscopy examination.     -   8. Prior, normal prostate examination (no palpable prostatic         mass) from the Nasobol-01-2009 trial.     -   9. A serum PSA 4.0 ng/mL.

9.3.2 Exclusion Criteria

-   -   1. Significant inter-current disease of any type, in particular         liver, kidney, or heart disease, any form of diabetes mellitus         or psychiatric illness.     -   2. Limitations in mobility, defined as having difficulty walking         two blocks on a level surface or climbing 10 steps     -   3. Hematocrit >54% at screening.     -   4. History of cancer, excluding skin cancer.     -   5. History of nasal surgery, specifically turbinoplasty,         septoplasty, rhinoplasty, “nose job”, or sinus surgery.     -   6. Subject with prior nasal fractures.     -   7. Subject with active allergies, such as rhinitis, rhinorrhea,         and nasal congestion.     -   8. Subject with mucosal inflammatory disorders, specifically         pemphigus, and Sjogren's syndrome.     -   9. Subject with sinus disease, specifically acute sinusitis,         chronic sinusitis, or allergic fungal sinusitis.     -   10. History of nasal disorders (e.g., polyposis, recurrent         epistaxis (>1 nose bleed per month), abuse of nasal         decongestants) or sleep apnea.     -   11. Subject using any form of intra-nasal medication delivery,         specifically nasal corticosteroids and oxymetazoline containing         nasal sprays (e.g., Dristan 12-Hour Nasal Spray).     -   12. History of severe adverse drug reaction or leucopenia.     -   13. History of abnormal bleeding tendencies or thrombophlebitis         unrelated to venipuncture or intravenous cannulation.     -   14. Positive test for Hepatitis B, Hepatitis C, or HIV.     -   15. History of asthma and on-going asthma treatment.     -   16. History of sleeping problems.     -   17. Smokers (>10 cigarettes per day).     -   18. Regular drinkers of more than four (4) units of alcohol         daily (1 unit=300 mL beer, 1 glass wine, 1 measure spirit) or         those that may have difficulty in abstaining from alcohol during         the 48 hours prior to the 24-hour blood sampling visit.     -   19. History of, or current evidence of, abuse of alcohol or any         drug substance, licit or illicit; or positive urine drug and         alcohol screen for drugs of abuse and alcohol.     -   20. Current treatment with androgens (e.g.,         Dehydroepiandrostenedione, Androstenedione) or anabolic steroids         (e.g., Testosterone, Dihydrotestosterone).     -   21. Treatment with Estrogens, GnRH antagonists, or Growth         Hormone, within previous 12 months.     -   22. Treatment with drugs which interfere with the metabolism of         Testosterone, such as Anastrozole, Clomiphene, Dutasteride,         Finasteride, Flutamide, Ketoconazole, Spironolactone and         Testolactone.     -   23. Androgen treatment within the past four weeks         (intramuscular, topical, buccal, etc.).     -   24. Subject with poor compliance history or unlikely to maintain         attendance.     -   25. Participation in any other research study during the conduct         of this study or 30 days prior to the initiation of this study,         with the exception of Nasobol-01-2009.     -   26. Blood donation (usually 550 mL) at any time during this         study, and within the 12 week period before the start of this         study.

9.3.3 Removal of Subjects from Therapy or Assessment

Subjects are informed that they are free to withdraw from the study at any time without having to give reasons for their withdrawal, and without consequences for their future medical care. They are asked to inform the investigator immediately of their decision. The subject's participation in the study may have been discontinued for any of the following reasons:

-   -   Subject's own wish.     -   Significant non compliance with the study protocol and         procedures.     -   Inter-current illness which interferes with the progress of the         study.     -   Intolerable adverse event, including clinically significant         abnormal laboratory findings, where, in the opinion of the         Clinical Investigator, these could interfere with the subject's         safety.     -   Clinical Investigator's decision that the withdrawal from the         study is in the best interest of the subject.

The Clinical Investigator had the right to terminate a study prematurely for safety reasons, after having informed and consulted with the Sponsor. The Sponsor had the right to terminate the study earlier if the clinical observations collected during the study suggested that it might not be justifiable to continue or for other reasons as described in the contract between Sponsor and the clinical sites (e.g., administrative, regulatory, etc.). However this is not necessary. There are no premature terminations or drops outs from the study.

9.4 Treatments

9.4.1 Treatments Administered

Subjects are centrally randomized to the following treatment groups in order to balance the numbers equally within the groups across the three centers:

-   -   Treatment A (n=10): TBS-1 syringes pre-filled with 125 μL 4.0%         gel to deliver 5.0 mg of Testosterone per nostril (intra-nasal)         given t.i.d. at 2100, 0700, and 1300 hours. (total dose 30         mg/day)     -   Treatment B (n=10): TBS-1 syringes pre-filled with 150 μL 4.5%         gel to deliver 6.75 mg of Testosterone per nostril (intra-nasal)         given b.i.d. at 2100 and 0700 hours. (total dose 27.0 mg/day)     -   Treatment C (n=10): TBS-1 syringes pre-filled with 125 μL 4.5%         gel to deliver 5.625 mg of Testosterone per nostril         (intra-nasal) given t.i.d. at 2100, 0700, and 1300 hours. (total         dose 33.75 mg/day)

9.4.2 Identity of Investigational Products

-   -   Name of the drug: TBS-1 (Syringes are pre-filled to contain 5.0         mg, 5.625 mg, and 6.75 mg of Testosterone/syringe).     -   Pharmaceutical form: Gel for nasal administration.     -   Content: Active ingredient: Testosterone.     -   Excipients: Silicon dioxide, castor oil, Labrafil®.     -   Mode of administration: Nasally, as a single dose to each         nostril.     -   Manufacturer: Haupt Pharma Amareg.     -   Batch numbers: 0744, 0942, and 0943     -   Storage conditions: Between 20-25° C.

Packaging

The TBS-1 study drug is delivered to the clinical trial site as a ready-for-use syringe in a foil pouch (two syringes per pouch). Examples of Syringe and Pouch Labels are described in Appendix 4 of the protocol.

9.4.3 Method of Assigning Subjects to Treatment

Subjects who met the entry criteria are assigned randomly on a 1:1:1 basis to one of the three treatment groups. At Screening, each subject is assigned a subject number by site in sequential order. Subject numbers consisted of 5 digits. The first 2 digits reflected the site number assigned to the investigator, followed by a 3-digit subject number. For example, 01-001 indicates site (01) and the first subject (001).

The subject number was used to identify the subject throughout the study and was entered on all documents. The same subject number was not assigned to more than one subject.

9.4.4 Selection of Doses in the Study

In a previous study, Nasobol-01-2009, a 3.2% Testosterone gel is used to deliver 4.0 mg, 5.5 mg and 7.0 mg of Testosterone intra-nasally using gel volumes of 125 μL, 172 μL and 219 μL, respectively. In this study, 5.0 mg, 5.65 mg and 6.75 mg of Testosterone are administered in gel volumes of 125 μL, 125 μL, and 150 μL, respectively. This study permits the investigation of the delivery of similar Testosterone amounts in much smaller volumes.

9.4.5 Selection and Timing of Dose for Each Subject

This was based on the results of the prior study.

9.4.6 Blinding

There is no blinding, because this is an open label study. The rationale for not blinding is that analytical endpoints, which are quantitative rather than qualitative are measured, and are not subject to any bias being introduced by the subjects or the Investigators.

9.4.7 Prior and Concomitant Therapy

The following medications are prohibited during the course of the study:

Subject using any form of intra-nasal medication delivery, specifically nasal corticosteroids and oxymetazoline containing nasal sprays (e.g., Dristan 12-Hour Nasal Spray).

Current treatment with androgens (e.g., Dehydroepiandrostenedione, Androstenedione) or anabolic steroids (e.g., Testosterone, Dihydrotestosterone). Treatment with Estrogens, GnRH antagonists, or Growth Hormone, within previous 12 months.

Treatment with drugs which interfere with the metabolism of Testosterone, such as; Anastrozole, Clomiphene, Dutasteride, Finasteride, Flutamide, Ketoconazole, Spironolactone and Testolactone.

Androgen treatment within the past four weeks (intramuscular, topical, buccal, etc.).

9.4.8 Treatment Compliance

All drugs are dispensed in accordance with the protocol. It is the Principal Investigator's responsibility to ensure that an accurate record of drugs issues and return is maintained. At the end of the study, the used original packages are returned to the sponsor for destruction. Drug accountability is verified by the monitors during the course of the study and prior to destruction of remaining study drugs.

During Visit 2, the subjects are given a one-week supply of pouches; 18 pouches for treatment A, 12 pouches for treatment B, and 18 pouches for treatment C. Each pouch contained two syringes prefilled with TBS-one gel for treatment A, B, or C. The subjects are instructed on how to administer the gel and are also given a diary to indicate the times of administration at their home.

9.5 Efficacy and Safety Variables

9.5.1 Efficacy and Safety Measurements Assessed

The primary efficacy parameter is the AUC is obtained in the 24 hours post administration of TBS-1. From the AUC the 24 hour C_(avg) is calculated.

-   -   Area under the concentration curve (AUC) for both b.i.d. and         t.i.d. dosing is determined for the 0 to 24 hour time interval         using the trapezoidal rule.     -   The average concentration in the dosing interval (C_(avg)) is         calculated from the AUC using the following formula:         C_(avg)=AUC_(0-τ)/τ, with τ=dosing interval time.     -   Peak Trough Fluctuation (PTF) and Peak Trough Swing (PTS) is         calculated as follows:

PTF=(C _(max) −C _(min))/C _(avg)

PTS=(C _(max) −C _(min))/C _(min)

-   -   C_(min), C_(max) and t_(max) is taken from the actual measured         values. Values are determined relative to the Testosterone         administration time in treated subjects.     -   The percent of subjects with 24 hour C_(avg) values for serum         Testosterone, DHT and Estradiol concentration above, within, and         below the respective reference range are calculated.     -   Additional exploratory analyses of PK parameters may have been         performed as necessary.

Analysis of Safety Data

Erythrocytosis, anemia, and infections are monitored by measuring complete blood counts at screening, and the Close-Out visit. An Otorhinolaryngological physician examined subjects and identifies any clinically significant changes to the nasal mucosa at follow up compared to baseline.

Clinical chemistry and urinalysis testing at Screening Visit 1 and at Close Out are assessed, hypo or hyperglycemia, renal function, liver function (hepato-cellular or obstructive liver disease), skeletal/heart muscle damage, and changes in calcium homeostasis.

Serum PSA is measured as a cautionary measure to measure possible changes to the prostate, although changes to the prostate and to serum PSA is not expected in a short treatment time frame.

Measurement of serum Testosterone, Dihydrotestosterone and Estradiol, at Screening Visit 1 and Visit 3 permitted any excursions beyond the upper limit of the reference range for the two physiological products of Testosterone; DHT, and Estradiol to be observed.

The safety analysis is performed on all subjects who received TBS-1. Occurrence of adverse events are presented by treatment group, by severity, and by relationship to the study drugs. All adverse events are described and evaluated regarding causality and severity. Adverse events are classified using MedDRA. However they are very few and all but two are not related to the drug.

Subject Safety

-   -   Monitoring of subjects and emergency procedures: Emergency         medication, equipment and Subject gurney are available at the         Study Center. During the “at home” phase, the subjects have an         emergency call number to be able to contact the Clinical         Investigator.     -   Adverse events are defined as any untoward medical occurrence in         a subject or clinical trial subject having administered a         medicinal product and which may or may not have a causal         relationship with this treatment. An adverse event can therefore         be any unfavorable and unintended sign, laboratory finding,         symptom or disease temporally associated with the use of an         investigational medicinal     -   product, whether considered related to it or not. Any         pre-existing condition during the clinical trial which is         worsened during the clinical study is to be considered an         adverse event.     -   An adverse reaction is defined as any untoward and unintended         response to an investigational product related to any dose         administered. All adverse reactions judged by either the         Clinical Investigator or the Sponsor to have reasonable causal         relationship to a medicinal product qualified as adverse         reactions. This is meant to convey in general that there is         evidence or an argument to suggest a causal relationship.     -   An unexpected adverse reaction is defined as an adverse         reaction, the nature, or severity of which is not consistent         with the applicable product information.     -   A serious adverse event or serious adverse reaction is defined         as any untoward medical occurrence or effect that, at any dose,         results in death, is life threatening, requires hospitalization         or prolongation of existing in-Subject hospitalization, results         in persistent or significant disability or incapacity, or is a         congenital anomaly or birth defect.     -   The observation period is extended from the time the subject         began the study medication through the end of Visit 3 for         hypogonadal subjects. AEs that are continuing at the end of the         study period are followed until the Investigator believed that         the AEs reached a stable clinical endpoint or are resolved.     -   The percent of subjects with a serum DHT and Estradiol greater         than the upper limit of the reference range, for the respective         analytes.     -   The Day 8 close-out findings are compared to the screening         results, and clinically significant changes identified in the         following:         -   Vital Signs and Adverse Events: Blood Pressure, Body             Temperature, Respiratory Rate, Heart Rate.         -   Otorhinolaryngological examination.         -   Complete Blood Count to evaluate changes in white blood             count, hemoglobin and hematocrit.         -   Clinical chemistry profile; Na/K, glucose, urea, creatinine,             calcium, phosphate, uric acid, total bilirubin, albumin,             AST, ALT, ALP, GGT, CK, and PSA.     -   Classifications:         -   A serious adverse event (SAE) or serious adverse reaction:             Defined as any untoward medical occurrence or effect that at             any dose; results in death, is life-threatening, requires             in-Subject hospitalization or prolongation of existing             in-Subject hospitalization, results in persistent or             significant disability or incapacity, is a congenital             anomaly or birth defect, a medically important condition,             i.e., the AE jeopardized the subject, or requires             intervention to prevent one of the outcomes listed above.         -   Non-serious AE: Any AE not meeting the SAE criteria.         -   Intensity: An adverse event/reaction is classified as Mild,             Moderate, or Severe.         -   Causality: The adverse event may be considered an adverse             reaction to an investigational medicinal product when a             “reasonable causal relationship” exists between the event             and the investigational product. The following degree of             causal relationship might be considered:             -   Definite: plausible temporal relationship with drug                 administration and withdrawal, and re-appears after drug                 re-start.             -   Probable: plausible temporal relationship with drug                 administration.             -   Possible: plausible temporal relationship with drug                 administration but can reasonably be associated to other                 factors.             -   Unlikely: does not have plausible temporal relationship                 with drug administration.             -   Unknown: no sufficient elements to establish a                 correlation with drug intake.             -   Not Related: cannot be correlated to the drug                 administration.     -   Procedure to be followed in the case of adverse events: All         adverse events detected by the Clinical Investigator are         recorded in the special section of the Case Report Form. Any         event that is classified as serious, regardless of causal         relationship, is to have been reported to the CRO and Sponsor         within 24 hours. There are no serious adverse events.

9.5.2 Appropriateness of Measurements

All measurements used in this study are standard indices of efficacy, PK and safety and are generally recognised as reliable, accurate and relevant.

9.5.3. Primary Efficacy Variable(s)

Pharmacokinetic profiles of serum Testosterone for subjects dosed in Treatments A, B, and C that have:

-   -   1. A 24 hour C_(avg) value >300 ng/dL and <1050 ng/dL.     -   2. The percent of subjects in each treatment group with a 24         hour C_(avg) less than, within and above the serum Testosterone         reference range of 300 ng/dL-1050 ng/dL.

9.6 Data Quality Assurance

The CRF entries are verified by the monitors against source documents. All entries into the database included the CRF and Diary Card subject data, the PK results, and laboratory values. All data is 100% audited after being entered into the database for this report.

9.7 Statistical Methods Planned in the Protocol and Determination of Sample Size

9.7.1 Statistical and Analytical Plans

The PK Analysis Plan is described above. The Analysis Plan for the Vital Signs and Laboratory Results are compared baseline results with final visit results after PK analysis. Other data including demographic data is descriptive. No statistical analysis is performed because group sizes are not selected on the basis of statistical significance.

9.7.2 Determination of Sample Size

Based on the results are obtained from conducting several pharmacokinetic studies in groups of 10 subjects per cohort, these are sufficient for an acceptable description of the pharmacokinetic parameters in this population. As this is a relatively modest Phase II PK study with the intent of investigating two higher concentrations of TBS-1 gel, a true sample size calculation is not performed.

9.8 Changes in the Conduct of the Study or Planned Analysis

The protocol is amended on Jul. 27, 2010. The change requested is in the timing of blood draws. The number of blood draws remained the same. This change is required to enable the full capture of the peak of testosterone absorption following the third TID dosing which occurred at 1300 hours on Day 8 or 1600 hours after the initial 2100 hour drug administration on the previous day (Day 7).

10. Study Subjects 10.1 Disposition of Subjects

The study is conducted at three centers located in Miami, Fla., Shreveport, L A and Tucson, Ariz.

The three treatment groups are equally divided amongst the three sites. Eight Subjects received Treatment A, seven Subjects received Treatments B and C, respectively. A total of 22 subjects are in the study. In addition, five subjects who participated in the previous clinical study failed screening and are therefore not randomized to the study.

TABLE 10.1 Disposition of Subjects by Site and Treatment Treatment A: Treatment B: Treatment C: TBS-1 syringe TBS-1 syringe TBS-1 syringe prefilled with prefilled with prefilled with SITE 125 micro-liters 150 micro- 125 micro- ID of drug liters of drug liters of drug Total 01 3 3 3 9 02 3 2 2 7 03 2 2 2 6 Total 8 7 7 22

10.2 Protocol Deviations

There are no meaningful pharmacokinetic deviations.

11. Pharmacokinetics and Statistics 11.1 Datasets Analyzed

The PK population is defined as subjects who receive the Treatment A, B or C, and who complete the study without major protocol violation or for whom the PK profile can be adequately characterized. The PK population is used for the analysis of PK data.

Based on the above criteria, twenty-two (22) subjects are included in the PK population. The numbers of subjects by site and by treatment are displayed below.

TABLE 11.1.1 Disposition of Subjects in the PK population: Site Number of Subjects 1 9 2 7 3 6 Treatment Number of Subjects A: TBS-1 125 μL of 4.0% Gel (t.i.d.) 8 B: TBS-1 150 μL of 4.5% Gel (b.i.d.) 7 C: TBS-1 125 μL of 4.5% Gel (t.i.d.) 7

11.2 Demographic and Other Baseline Characteristics

The demographic data and characteristics are presented by dose group for all the treated subjects in Table 11.2. No meaningful differences are observed amongst the three groups for any of the characteristics.

TABLE 11.2 Summary of Demographic Characteristics-All Subjects Treatment A: Treatment B: Treatment C: TBS-1 TBS-1 TBS-1 syringe syringe syringe prefilled with prefilled with prefilled with 125 micro- 150 micro- 125 micro- liters of 4.0 liters of 4.5 liters of 4.5 All percent gel percent gel percent gel Subjects Characteristic (N = 8) (N = 7) (N = 7) (N = 22) SEX Male 8 7 7 22 RACE Black or 1 1 African American White 8 7 6 21 ETHNIC Hispanic or 4 3 3 10 Latino Non-Hispanic 4 4 4 12 and Non-Latino AGE Mean 52.38 53.86 51.57 52.59 SD 12.55 11.04 9.90 10.78 Minimum 37 36 35 35 Maximum 73 63 67 73 Median 51 59 52 54

The treated populations for Group A have a mean age of 52.38, for Group B 53.86, and for Group C 51.57. The standard deviations are 12.55, 11.04, and 9.90, respectively. The ethnic and racial distribution are essentially the same in each group.

11.3 Measurement of Treatments Compliance

Compliance of drug utilization during the home portion of the study is determined by a review of the diaries and used returned pouches and syringes. Although the method is not absolute, it is sufficient to establish reasonable compliance. One subject could not find his diary.

11.4 Pharmacokinetics and Statistical Results

11.4.1 Methods

The blood concentrations are received from ABL and transferred electronically from Trimel Biopharma SRL to the statistical unit of PharmaNet. Testosterone and Dihydrotestosterone serum concentrations are provided in ng/mL. However, the serum concentrations are converted to ng/dL for PK calculation to match the units of the literature's reference ranges.

During the trial, clinical site 1 performs PK sampling one day later than specified in the protocol that is it started on Day 8 rather than Day 7. This change is not planned. Consequently, the actual times are calculated relative to the 2100 drug administration on Day 8 for the subjects of clinical site 1 and the drug administration 21 h00 on Day 7 for the subjects of clinical sites 2 and 3.

For subject No. 02-003, the dosing time is not recorded on Day 7. Consequently, the schedule sampling times are used instead of the actual sampling times for PK calculations. The 16.33 h and 16.67 h samples for subject 01-001 are drawn at the same time due to technical reason. The schedule sampling time is used for sample 16.33 h while the actual sampling time is used for sample 16.67 h.

Excluding the above exceptions, time deviations during sampling are treated as follows: for all sampling times, the difference between the scheduled and the actual sampling time is considered acceptable if it is less than 1 minute. When the difference exceeded this time limit, the actual sampling times (rounded off to three decimal digits) are used to calculate pharmacokinetic parameters, except for pre-dose samples, which are always reported as zero (0.000), regardless of time deviations. Scheduled sampling times are presented in concentration tables and graphs in the statistical report.

PK calculations are performed using WinNonlin™ version 5.2 (or higher), validated according to industry's expectations and regulatory requirements. Descriptive statistical calculations are also performed using Microsoft® Office Excel 2003. Microsoft® Office Excel 2003 and Microsoft® Office Word 2003 are used for report data tabulation.

Descriptive statistics (N, mean, standard deviation (SD), coefficient of variation (CV), median, minimum value (Min.), and maximum value (Min.)) of the serum concentrations versus time as well as all pharmacokinetic parameters are provided for each treatment at each dose level using the evaluable population. All figures are presented using both linear (a) and semi-log (b) scales.

For the calculation of the PK parameters from the last three drug administrations (Treatments A and C: 0 hour to 10 hours, 10 hours and 16 hours and 16 hours and 24 hours; treatment B: 0 hour to 10 hours and 10 hours and 24 hours), the serum concentration values for Testosterone, Dihydrotestosterone, and Estradiol at time points 10 hours (pre-dose for the second drug administration) and 16 hours (pre-dose for the third drug administration under Treatments A and C) are obtained by imputing the serum concentration value observed at time points 9.75 hours and 15.75 hours, respectively.

The following pharmacokinetic parameters are determined for all subjects for Testosterone, Dihydrotestosterone and Estradiol:

For Treatments A and C (t.i.d.): AUC_(0-τ), AUC₀₋₁₀, AUC₁₀₋₁₆, AUC₁₆₋₂₄, C_(max), C_(max 0-10), C_(max 10-16), C_(max 16-24), C_(min), C_(min 0-10), C_(min 10-16), C_(min 16-24), C_(avg), C_(avg 0-10), C_(avg 10-16), C_(avg 16-24), t_(max), t_(max 0-10), t_(max 10-16), t_(max 16-24), t_(max 10-24), PTF, PTS.

For Treatment B (b.i.d.): AUC_(0-τ), AUC₀₋₁₀, AUC₁₀₋₂₄, C_(max), C_(max 0-10), C_(max 10-24), C_(min), C_(min 0-10), C_(min 10-24), C_(avg), C_(avg 0-10), C_(avg 10-24), t_(max), t_(max 0-10), t_(max 10-24), PTF, PTS.

Additionally, the percent of subjects with C_(avg) values for serum Testosterone, Dihydrotestosterone and Estradiol above, within, and below their respective reference range is calculated for each treatment. As well, the mean percent time of serum Testosterone, Dihydrotestosterone and Estradiol values above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the corresponding reference range are provided for each treatment. The calculation of all these pharmacokinetic parameters is explained below.

11.4.1.1 Maximum and Minimum Observed Concentrations and Time of Observed Peak Concentrations

C_(max), the maximum is observed concentrations and T_(max), the time to reach that peak concentrations, as well as C_(min), the minimum observed concentrations are determined for each subject and for each treatment as follow:

-   C_(max): Maximum observed concentration over the dosing interval.     This parameter is calculated for Treatments A, B and C. -   C_(max 0-10): Maximum observed concentration from time zero to 10     hours. This parameter is calculated for Treatments A, B and C. -   C_(max 10-16): Maximum observed concentration from time 10 hours to     16 hours. This parameter is calculated for Treatments A and C. -   C_(max 16-24): Maximum observed concentration from time 16 hours to     24 hours. This parameter is calculated for Treatments A and C. -   C_(max 10-24): Maximum observed concentration from time 10 hours to     24 hours. This parameter is calculated for Treatment B only. -   C_(min): Minimum observed concentration over the dosing interval.     This parameter is calculated for Treatments A, B and C. -   C_(min 0-10): Minimum observed concentration from time zero to 10     hours. This parameter is calculated for Treatments A, B and C. -   C_(min 10-16): Minimum observed concentration from time 10 hours to     16 hours. This parameter is calculated for Treatments A and C. -   C_(min 16-24): Minimum observed concentration from time 16 hours to     24 hours. This parameter is calculated for Treatments A and C. -   C_(min 10-24): Minimum observed concentration from time 10 hours to     24 hours. This parameter is calculated for Treatment B only. -   t_(max): Time of observed C_(max) over the dosing interval. This     parameter is calculated for Treatments A, B and C. -   t_(max 0-10): Time of observed C_(max) from time zero to 10 hours.     This parameter is calculated for Treatments A, B and C. -   t_(max 10-16): Time of observed C_(max) from time 10 hours to 16     hours. This parameter is calculated for Treatments A and C. -   t_(max 16-24): Time of observed C_(max) from time 16 hours to 24     hours. This parameter is calculated for Treatments A and C. -   t_(max 10-24): Time of observed C_(max) from time 10 hours to 24     hours. This parameter is calculated for Treatment B only.

11.4.1.2 Areas Under the Concentration-Time Curves

The calculation of AUCs is performed using the linear trapezoidal method. AUC_(0-τ) is computed from dose time (0) to dose time □ (□=24 h). However, in case the 24-h sample is collected with a time deviation, the AUC₀₋τ is estimated based on the estimated concentration at 24 hours using the regression line calculated from the elimination phase, and not the concentration at the actual observation time.

In the case where the last concentration value (Y) is missing or does not correspond to a scheduled sampling time (i.e. 10 hours and 16 hours), AUC_(X-Y) is extrapolated using the corresponding subject's elimination phase, if calculable.

The following AUCs are calculated:

-   AUC_(0-τ): Area under the concentration-time curve for one dosing     interval. This parameter is calculated for Treatments A, B and C. -   AUC₀₋₁₀: Area under the concentration-time curve from time zero to     10 hours. This parameter is calculated for Treatments A, B and C. -   AUC₁₀₋₁₆: Area under the concentration-time curve from time 10 hours     to 16 hours. This parameter is calculated for Treatments A and C. -   AUC₁₆₋₂₄: Area under the concentration-time curve from time 16 hours     to 24 hours. This parameter is calculated for Treatments A and C. -   AUC₁₀₋₂₄: Area under the concentration-time curve from time 10 hours     to 24 hours. This parameter is calculated for Treatment B only.     The C_(avg) are calculated as follow: -   C_(avg): Average concentration during the dosing interval,     calculated as AUC₀-τ/τ (τ=24 hours). This parameter is calculated     for Treatments A, B and C. -   C_(avg 0-10): Average concentration from time zero to 10 hours,     calculated as AUC0-10/10. This parameter is calculated for     Treatments A, B and C. -   C_(avg 10-16): Average concentration from time 10 hours to 16 hours,     calculated as AUC10-16/6. This parameter is calculated for     Treatments A and C. -   C_(avg 16-24): Average concentration from time 16 hours to 24 hours,     calculated as AUC16-24/8. This parameter is calculated for     Treatments A and C. -   C_(avg 10-24): Average concentration from time 10 hours to 24 hours,     calculated as AUC10-24/14. This parameter is calculated for     Treatment B only.

11.4.1.3 Average Drug Concentrations

The C_(avg) are calculated as follow:

-   C_(avg): Average concentration during the dosing interval,     calculated as AUC0-τ/τ (τ=24 hours). This parameter is calculated     for Treatments A, B and C. -   C_(avg 0-10): Average concentration from time zero to 10 hours,     calculated as AUC0-10/10. This parameter is calculated for     Treatments A, B and C. -   C_(avg 10-16): Average concentration from time 10 hours to 16 hours,     calculated as AUC10-16/6. This parameter is calculated for     Treatments A and C. -   C_(avg 16-24): Average concentration from time 16 hours to 24 hours,     calculated as AUC16-24/8. This parameter is calculated for     Treatments A and C. -   C_(avg 10-24): Average concentration from time 10 hours to 24 hours,     calculated as AUC10-24/14. This parameter is calculated for     Treatment B only.

11.4.1.4 Peak Trough Fluctuation and Peak Trough Swing

The peak trough fluctuation (PTF) and the Peak trough swing are calculated as follow:

-   PTF: Peak trough fluctuation, calculated as     (C_(max)−C_(min))/C_(avg) This parameter is calculated for     Treatments A, B and C. -   PTS: Peak trough swing, calculated as (C_(max)−C_(min))/C_(min).     This parameter is calculated for Treatments A, B and C.

11.4.1.5 Percent Time Above, within and Below the Reference Range and Percent of a Subjects with C_(avg) Above, within and Below the Reference Range

The percent times during which observations fall above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the reference ranges are computed for each subject and treatment for the serum Testosterone, Dihydrotestosterone and Estradiol. The percent of subjects with C_(avg) values for serum Testosterone, Dihydrotestosterone and Estradiol above, within, and below their respective reference range is calculated for each treatment. The reference ranges are 300 ng/dL to 1050 ng/dL for Testosterone, 25.5 ng/dL to 97.8 ng/dL for Dihydrotestosterone and 3 pg/mL to 81 pg/mL for Estradiol.

-   -   PTS: Peak trough swing, calculated as (C_(max)−C_(min))/C_(min).         This parameter is calculated for Treatments A, B and C.

11.4.1.6 Statistical Analysis

Only descriptive statistics (N, mean, SD, CV, median, Min., and Max.) are calculated on the serum concentrations and the PK parameters for each treatment. No inferential statistical analysis is performed.

11.4.2 Analysis of Pharmacokinetics and Statistical Issues

11.4.2.2 Handling of Missing Data

Samples that are not analyzed due to an insufficient volume (refer to the bioanalytical report) are recorded as INV (Insufficient volume for analysis) in the concentration tables.

These samples are set as missing for pharmacokinetic and statistical analyses. As the PK parameters could be estimated using the remaining data points, subjects with missing data are kept in the pharmacokinetic analysis.

11.4.2.3 Pharmacokinetic Analysis

The following pharmacokinetic parameters are determined for all subjects for

Testosterone, Dihydrotestosterone and Estradiol:

For Treatments A and C AUC_(0-τ), AUC₀₋₁₀, AUC₁₀₋₁₆, AUC₁₆₋₂₄, C_(max), C_(max 0-10), C_(max 10-16), C_(max 16-24), C_(min), C_(min 0-10), C_(min 10-16), C_(min 16-24), C_(avg), C_(avg 0-10), C_(avg 10-16), C_(avg 16-24), t_(max), t_(max 0-10), t_(max 10-16), t_(max 16-24), t_(max 10-24), PTF, PTS.

For Treatment B (b.i.d.): AUC_(0-τ), AUC₀₋₁₀, AUC₁₀₋₂₄, C_(max), C_(max 0-10), C_(max 10-24), C_(min), C_(min 0-10), C_(min 10-24), C_(avg), C_(avg 0-10), C_(avg 10-24), t_(max), t_(max 0-10), t_(max 10-24), PTF, PTS.

Additionally, the percent of subjects with C_(avg) values for serum Testosterone, Dihydrotestosterone and Estradiol above, within, and below their respective reference range is calculated for each treatment. As well, the mean percent time of serum Testosterone, Dihydrotestosterone and Estradiol values above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the corresponding reference range are provided for each treatment. The calculation of all these pharmacokinetic parameters is explained below.

With the exception of text Tables (numbered as 11.4.2.3-1 to 11.4.2.3-3) and text Figures (numbered as 11.4.2.3-1 to 11.4.2.3-3), all tables and figures referred to in this section are displayed in sections 14.2.1 and 14.2.2, respectively. For brevity, TBS-1 treatments are identified in the text of the statistical report by their treatment code: A (125 μL of 4% gel given t.i.d. for a total dose of 30 mg/day), B (150 μL of 4.5% gel is given b.i.d. for a total dose of 27.0 mg/day) and C (125 μL of 4.5% gel given t.i.d. for a total dose of 33.75 mg/day).

Blood samples for pharmacokinetic analysis are collected prior and post the 2100 hour drug administration on Day 7 at 0.333, 0.667, 1.00, 1.50, 2.00, 3.00, 6.00, 9.00, 9.75, 10.33, 10.66, 11.0, 11.5, 12.0, 13.0, 14.0, 15.75, 16.33, 16.66, 17.0, 17.5, 18.0, 20.0, 22.0, and 24.0 hours for Treatments A and C. Blood samples for pharmacokinetic analysis are collected prior and post the 2100 hour drug administration on Day 7 at 0.333, 0.667, 1.00, 1.50, 2.00, 3.00, 6.00, 9.00, 9.75, 10.33, 10.66, 11.0, 11.5, 12.0, 13.0, 16.0, 19.0, 22.0, and 24.0 hours for Treatment B. The actual sampling times is used for PK calculation are displayed in Tables 14.2.1.22, 14.2.1.23 and 14.2.1.24 for Treatments A, B and C, respectively.

Testosterone

The Testosterone serum concentrations measured for each subject at each sampling time appear in Tables 14.2.1.1, 14.2.1.2 and 14.2.1.3 according to treatment. The plots of the individual serum levels over the sampling period are presented using both linear (a) and semi-log (b) scales in FIGS. 14.2.2.1 through 14.2.2.22. Lines for the minimum (300 ng/dL) and maximum (1050 ng/dL) bound of the reference range for the testosterone serum concentrations are also presented for information purposes. As well, a line for the average drug concentration (C_(avg)) during the dosing interval (τ=24 hours) is also presented on the individual profiles.

The plots of the mean serum levels over the sampling period are also presented using both the linear (a) and semi-log (b) scales in FIGS. 14.2.2.23, 14.2.2.24 and 14.2.2.25 for Treatments A, B and C, respectively. The error bars on these mean profiles correspond to one standard deviation. The lines for the minimum and maximum bound of the reference ranges are also presented on the mean figures.

The mean plot on the linear scale for each treatment is also presented below in the text FIG. 11.4.2.3-1.

As shown in FIG. 35 the mean testosterone serum concentration (ng/dL) Time Profile for Each Treatment.

Calculated pharmacokinetic parameters for each subject according to treatment are shown in Tables 14.2.1.4, 14.2.1.5 and 14.2.1.6 for Treatments A, B and C, respectively. They are summarized in the text Table 11.4.2.3-1.

TABLE 11.4.2.3-1 Summary of Testosterone Pharmacokinetic Parameters for Each Treatment Treatment A¹ (N = 8) Treatment B² (N = 7) Treatment C³ (N = 7) Parameter Unit Mean SD CV % Mean SD CV % Mean SD CV % AUC₀₋₁₀ h * ng/dL 4178.68 1210.51 28.97 4451.64 1581.09 35.52 4355.19 1374.07 31.55 C_(max 0-10) ng/dL 786 209 26.53 894 500 55.90 857 323 37.72 C_(min 0-10) ng/dL 259 70.3 27.16 256 91.5 35.76 272 69.7 25.61 C_(avg 0-10) ng/dL 418 121 28.97 445 158 35.52 436 137 31.55 T_(max 0-10) h 1.01 0.678 67.21 0.695 0.279 40.18 0.905 0.422 46.62 AUC₁₀₋₁₆ h * ng/dL 2635.05 1062.56 40.32 — — — 2301.51 658.44 28.61 C_(max 10-16) ng/dL 698 251 35.88 — — — 675 256 37.98 C_(min 10-16) ng/dL 270 90.7 33.63 — — — 230 53.9 23.48 C_(avg 10-16) ng/dL 439 177 40.32 — — — 384 110 28.61 T_(max 10-16) h 11.1 1.06 9.54 — — — 10.8 0.562 5.20 AUC₁₀₋₂₄ h * ng/dL — — — 5264.19 2176.63 41.35 — — — C_(max 10-24) ng/dL — — — 846 377 44.53 — — — C_(min 10-24) ng/dL — — — 228 100 43.88 — — — C_(avg 10-24) ng/dL — — — 376 155 41.35 — — — T_(max 10-24) h — — — 11.1 0.675 6.06 — — — AUC₁₆₋₂₄ h * ng/dL 3016.52 1083.58 35.92 2766.97 838.13 30.29 C_(max 16-24) ng/dL 556 216 38.78 — — — 595 352 59.20 C_(min 16-24) ng/dL 271 86.9 32.08 — — — 225 59.1 26.26 C_(avg 16-24) ng/dL 377 135 35.92 — — — 346 105 30.29 T_(max 16-24) h 16.6 0.404 2.43 — — — 16.8 0.704 4.19 AUC₀₋— h * ng/dL 9920.07 3300.65 33.27 9781.39 3532.43 36.11 9505.03 2650.59 27.89 C_(max) ng/dL 830 188 22.65 1050 463 44.19 883 346 39.23 C_(min) ng/dL 239 77.6 32.55 224 98.6 43.97 222 57.1 25.69 C_(avg) ng/dL 413 138 33.27 408 147 36.11 396 110 27.89 T_(max) h 4.61 5.27 114.31 4.99 5.43 108.81 4.50 6.44 143.18 PTF — 1.51 0.39 26.03 2.04 1.07 52.23 1.61 0.47 28.92 PTS — 2.63 0.73 27.70 4.49 3.92 87.27 3.04 1.65 54.27 % % 34.47 30.93 89.72 36.40 25.92 71.22 30.14 29.25 97.05 TimeBelow * % % 65.16 30.46 46.75 59.47 23.10 38.84 68.21 28.77 42.17 TimeWithin % * % 0.38 1.06 282.84 4.13 6.88 166.67 1.65 2.60 157.31 TimeAbove C_(avg) Below * % 1 — — 1 — — 1 — — [N (% of (12.50%) (14.29%) (14.29%) Subjects) C_(avg) Within * % 7 — — 6 — — 6 — — [N (% of (87.50%) (85.71%) (85.71%) Subjects) C_(avg) Above * % 0 — — 0 — — 0 — — [N (% of (0%)   (0%)   (0%)   Subjects)] *Reference Range = 300-1050 ng/dL. ¹TBS-1, 125 μL 4.0% gel given t.i.d. (total dose 30 mg/day) ²TBS-1, 150 μL of 4.5% gel given b.i.d. (total dose 27.0 mg/day) ³TBS-1, 125 μL of 4.5% gel given t.i.d. (total dose 33.75 mg/day)

The percent times during which observations fall above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the reference range are computed for each subject and are presented in Tables 14.2.1.4, 14.2.1.5 and 14.2.1.6 for Treatments A, B and C, respectively. These results are also summarized in text Table 11.4.2.3.1.

The percent of subjects with C_(avg) values for serum Testosterone above, within, and below the reference range is calculated for each treatment and are presented in Table 14.2.1.7. These results are also summarized in text Table 11.4.2.3.1.

Dihydrotestosterone

The Dihydrotestosterone serum concentrations are measured for each subject at each sampling time appear in Tables 14.2.1.8, 14.2.1.9 and 14.2.1.10 according to treatment. The plots of the individual serum levels over the sampling period are presented using both linear (a) and semi-log (b) scales in FIGS. 14.2.2.26 through 14.2.2.47. Lines for the minimum (25.5 ng/dL) and maximum (97.8 ng/dL) bound of the reference range for the Dihydrotestosterone serum concentrations are also presented for information purposes. As well, a line for the average drug concentration (C_(avg)) during the dosing interval (τ=24 hours) is also presented on the individual profiles.

The plots of the mean serum levels over the sampling period are also presented using both the linear (a) and semi-log (b) scales in FIGS. 14.2.2.48, 14.2.2.49 and 14.2.2.50 for Treatments A, B and C, respectively. The error bars on these mean profiles correspond to one standard deviation. The lines for the minimum and maximum bound of the reference ranges are also presented on the mean figures.

The mean plot on the linear scale for each treatment is also presented below in the text FIG. 11.4.2.3-2.

As shown in FIG. 36 depicts the Mean Dihydrotestosterone Serum Concentration (ng/dL) Time Profile for Each Treatment is depicted.

As per SAP, AUC_(X-Y) is calculated based on the estimated concentration (Y) using the regression line calculated from the elimination phase data when the last concentration (Y) does not correspond to a schedule sampling time. For subject No. 01-002 and 02-007, the elimination phase is not well characterized due to fluctuation in the Dihydrotestosterone serum concentration for the 10 to 16 hours and 0 to 10 hours intervals, respectively. Therefore, AUC₁₀₋₁₆ and C_(avg 10-16) (derived from AUC₁₀₋₁₆) could not be calculated for subject No. 01-002 for Treatment A (N=7 for these parameters). As well, AUC₀₋₁₀ and C_(avg 0-10) (derived from AUC₀₋₁₀) could not be calculated for subject No. 02-007 for Treatment A (N=7 for these parameters).

Calculated pharmacokinetic parameters for each subject according to treatment are shown in Tables 14.2.1.11, 14.2.1.12 and 14.2.1.13 for Treatments A, B and C, respectively. They are summarized in the text Table 11.4.2.3-2.

TABLE 11.4.2.3-2 Summary of Dihydrotestosterone Pharmacokinetic Parameters for Each Treatment Treatment A¹ Treatment B² Treatment C³ (N = 8) (N = 7) (N = 7) Parameter Unit Mean SD CV % Mean SD CV % Mean SD CV % AUC₀₋₁₀ ^(a) h * ng/dL 345.77 133.49 38.61 402.77 133.11 33.05 411.10 131.22 31.92 C_(max 0-10) ng/dL 51.4 18.8 36.52 56.8 17.1 30.08 59.0 19.7 33.48 C_(min 0-10) ng/dL 26.6 10.1 38.15 30.1 13.4 44.57 31.7 9.33 29.41 C_(avg 0-10) ^(a) ng/dL 34.6 13.3 38.61 40.3 13.3 33.05 41.1 13.1 31.92 T_(max 0-10) h 2.38 2.98 125.22 1.70 0.501 29.48 1.32 0.569 43.20 AUC₁₀₋₁₆ ^(a) h * ng/dL 186.33 65.10 34.94 — — — 222.62 53.52 24.04 C_(max 10-16) ng/dL 44.2 16.8 38.01 — — — 48.9 12.4 25.37 C_(min 10-16) ng/dL 26.6 10.4 38.95 — — — 30.1 8.41 27.94 C_(avg 10-16) ng/dL 31.1 10.8 34.94 — — — 37.1 8.92 24.04 T_(max 10-16) h 11.9 1.13 9.50 — — — 11.4 0.436 3.84 AUC₁₀₋₂₄ h * ng/dL — — — 543.29 235.71 43.39 — — — C_(max 10-24) ng/dL — — — 54.6 21.9 40.12 — — — C_(min 10-24) ng/dL — — — 28.3 12.7 45.02 — — — C_(avg 10-24) ng/dL — — — 38.8 16.8 43.39 — — — T_(max 10-24) h — — — 11.8 0.775 6.55 — — — AUC₁₆₋₂₄ h * ng/dL 269.16 114.13 42.40 — — — 275.21 74.02 26.89 C_(max 16-24) ng/dL 41.3 17.0 41.20 — — — 42.6 12.8 30.15 C_(min 16-24) ng/dL 26.5 11.3 42.63 — — — 26.6 6.41 24.11 C_(avg 16-24) ng/dL 33.6 14.3 42.40 — — — 34.4 9.25 26.89 T_(max 16-24) h 17.6 1.37 7.79 — — — 17.5 0.433 2.48 AUC_(0-τ) h * ng/dL 818.95 315.07 38.47 946.89 361.03 38.13 909.68 249.37 27.41 C_(max) ng/dL 52.2 18.1 34.64 61.0 22.5 36.85 60.3 18.6 30.84 C_(min) ng/dL 25.3 10.1 40.14 27.8 13.0 46.69 26.6 6.41 24.11 C_(avg) ng/dL 34.1 13.1 38.47 39.5 15.0 38.13 37.9 10.4 27.41 T_(max) h 4.43 6.01 135.63 4.42 4.84 109.53 4.26 5.18 121.44 PTF — 0.82 0.28 34.18 0.89 0.33 36.71 0.88 0.17 19.17 PTS — 1.14 0.44 39.02 1.36 0.70 51.43 1.24 0.30 23.90 % TimeBelow * % 32.64 35.13 107.62 26.22 30.06 114.63 13.87 36.41 262.41 % TimeWithin * % 67.36 35.13 52.15 73.78 30.06 40.74 86.13 36.41 42.27 % TimeAbove * % 0.0 0.00 — 0.0 0.000 — 0.0 0.000 — C_(avg) Below * % 3 — — 1 — — 1 — — [N (% of Subjects)] (37.50%) (14.29%) (14.29%) C_(avg) Within * 5 — — 6 — — 6 — — [N (% of Subjects)] % (62.50%) (85.71%) (85.71%) C_(avg) Above * % 0 — — 0 — — 0 — — [N (% of Subjects)] (0%)   (0%)   (0%)   *Reference Range = 25.5-97.8 ng/dL. ¹TBS-1, 125 μL 4.0% gel given t.i.d. (total dose 30 mg/day) ²TBS-1, 150 μL of 4.5% gel given b.i.d. (total dose 27.0 mg/day) ³TBS-1, 125 μL of 4.5% gel given t.i.d. (total dose 33.75 mg/day) ^(a)For these parameters, N = 7 for Treatment A.

The percent times during which observations fall above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the reference range are computed for each subject and are presented in Tables 14.2.1.11, 14.2.1.12 and 14.2.1.13 for Treatments A, B and C, respectively. These results are also summarized in text Table 11.4.2.3.2. The percent of subjects with C_(avg) values for serum Dihydrotestosterone above, within, and below the reference range is calculated for each treatment and are presented in Table 14.2.1.14. These results are also summarized in text Table 11.4.2.3.2.

Estradiol

The Estradiol serum concentrations are measured for each subject at each sampling time appear in Tables 14.2.1.15, 14.2.1.16 and 14.2.1.17 according to treatment. The plots of the individual serum levels over the sampling period are presented using both linear (a) and semi-log (b) scales in FIGS. 14.2.2.51 through 14.2.2.72. Lines for the minimum (3 pg/mL) and maximum (81 pg/mL) bound of the reference range for the Estradiol serum concentrations are also presented for information purposes. As well, a line for the average drug concentration (C_(avg)) during the dosing interval (τ=24 hours) is also presented on the individual profiles.

The plots of the mean serum levels over the sampling period are also presented using both the linear (a) and semi-log (b) scales in FIGS. 14.2.2.73, 14.2.2.74 and 14.2.2.75 for Treatments A, B and C, respectively. The error bars on these mean profiles correspond to one standard deviation. The lines for the minimum and maximum bound of the reference ranges are also presented on the mean figures.

The mean plot on the linear scale for each treatment is also presented below in the text FIG. 11.4.2.3-3.

As shown in FIG. 37 the mean estradiol serum concentration (pg/mL) Time Profile for Each Treatment is depicted.

As per SAP (section 8.3), AUC_(X-Y) is calculated based on the estimated concentration (Y) using the regression line calculated from the elimination phase data when the last concentration (Y) does not correspond to a schedule sampling time. However, for some subjects the elimination phase is not well characterized due to fluctuation in the Estradiol serum concentration as follows:

-   -   Subject No.: 02-007 for the 0 to 10 hours and for the 0 to 24         hours time intervals for Treatment A. The following PK         parameters could not be calculated for this subject: AUC₀₋₁₀,         C_(avg 0-10), AUC_(0-τ), C_(avg) and PTF for Treatment A (N=7         for these parameters).     -   Subject Nos: 01-002 and 01-007 for the 10 to 16 hours time         interval for Treatment A. The AUC₁₀₋₁₆ and C_(avg 10-16) could         not be calculated for these subjects for Treatment A (N=6 for         these parameters).     -   Subject Nos. 02-004 and 02-007 for the 16 to 24 hours time         interval for Treatment A. The AUC₁₆₋₂₄ and C_(avg 16-24) could         not be calculated for this subject for Treatment A (N=6 for         these parameters).     -   Subject Nos. 02-003 and 02-005 for the 0 to 10 hours time         interval for Treatment C. The AUC₀₋₁₀ and C_(avg 0-10) could not         be calculated for these subjects for Treatment C (N=5 for these         parameters).

Calculated pharmacokinetic parameters for each subject according to treatment are shown in Tables 14.2.1.18, 14.2.1.19 and 14.2.1.20 for Treatments A, B and C, respectively. They are summarized in the text Table 11.4.2.3-3.

TABLE 11.4.2.3-3 Summary of Estradiol Pharmacokinetic Parameters for Each Treatment Treatment A¹ (N = 8) Treatment B² (N = 7) Treatment C³ (N = 7) Parameter Unit Mean SD CV % Mean SD CV % Mean SD CV % AUC₀₋₁₀ ^(b, c) h * pg/mL 234.96 95.96 40.84 242.02 64.26 26.55 267.78 75.37 28.15 C_(max 0-10) pg/mL 36.8 13.4 36.33 35.8 9.06 25.29 35.5 7.75 21.80 C_(min 0-10) pg/mL 17.7 6.43 36.35 17.4 5.67 32.63 22.1 8.07 36.43 C_(avg 0-10) ^(b, c) pg/mL 23.5 9.60 40.84 24.2 6.43 26.55 26.8 7.54 28.15 T_(max 0-10) h 2.62 2.87 109.67 1.49 0.608 40.85 2.68 3.38 126.14 AUC₁₀₋₁₆ ^(d) h * pg/mL 144.76 51.60 35.65 — — — 144.30 53.70 37.21 C_(max 10-16) pg/mL 28.9 10.8 37.29 — — — 31.5 8.82 28.02 C_(min 10-16) pg/mL 16.3 5.42 33.32 — — — 19.2 8.62 45.02 C_(avg 10-16) ^(d) pg/mL 24.1 8.60 35.65 — — — 24.0 8.95 37.21 T_(max 10-16) h 12.1 1.15 9.49 — — — 11.2 0.693 6.19 AUC₁₀₋₂₄ h * pg/mL — — — 295.12 81.19 27.51 — — — C_(max 10-24) pg/mL — — — 30.6 8.16 26.70 — — — C_(min 10-24) pg/mL — — — 15.9 4.46 27.95 — — — C_(avg 10-24) pg/mL — — — 21.1 5.80 27.51 — — — T_(max 10-24) h — — — 12.4 1.74 14.00 — — — AUC₁₆₋₂₄ ^(d) h * pg/mL 153.02 42.87 28.02 — — — 177.97 48.79 27.41 C_(max 16-24) pg/mL 27.2 10.4 38.23 — — — 26.9 7.99 29.74 C_(min 16-24) pg/mL 17.4 5.75 33.11 — — — 17.0 5.65 33.28 C_(avg 16-24) ^(d) pg/mL 19.1 5.36 28.02 — — — 22.2 6.10 27.41 T_(max 16-24) h 18.81 1.88 10.01 — — — 18.5 1.92 10.36 AUC_(0-T) ^(b) h * pg/mL 530.27 196.8 37.12 537.16 137.99 25.69 601.91 188.18 1.26 C_(max) pg/mL 37.9 13.6 35.97 36.2 8.69 24.04 36.4 8.44 23.18 C_(min) pg/mL 16.1 5.36 33.31 15.7 4.40 28.03 17.0 5.65 33.28 C_(avg) ^(b) pg/mL 22.1 8.20 37.12 22.4 5.75 25.69 25.1 7.84 31.26 T_(max) h 4.13 7.13 172.74 4.51 5.25 116.25 4.88 5.27 107.94 PTF^(b) — 0.97 0.35 36.08 0.93 0.28 30.25 0.81 0.21 25.16 PTS — 1.36 0.48 35.44 1.35 0.49 35.88 1.21 0.31 25.44 % % 0.00 0.00 — 0.00 0.00 — 0.00 0.00 — TimeBelow * % % 100.00 0.00 0.00 100.00 0.00 0.00 100.00 0.00 0.00 TimeWithin * % % 0.00 0.00 — 0.00 0.00 — 0.00 0.00 — TimeAbove * C_(avg) Below ^(b,) * % 0 (0%) — — 0 (0%) — — 0 (0%) — — [N (% of Subjects)] C_(avg) Within ^(b,) * % 7 — — 7 — — 7 — — [N (% of (100.00%) (100.00%) (100.00%) Subjects)] C_(avg) % 0 (0%) — — 0 (0%) — — 0 (0%) — — Above ^(b,) * [N (% of Subjects)] * Reference Range = 3-81 μg/mL. ¹TBS-1, 125 μL 4.0% gel given t.i.d. (total dose 30 mg/day) ²TBS-1, 150 μL of 4.5% gel given b.i.d. (total dose 27.0 mg/day) ³TBS-1, 125 μL of 4.5% gel given t.i.d. (total dose 33.75 mg/day) ^(b)or these parameters, N = 7 for Treatment A. ^(c)For these parameters, N = 6 for Treatment A. ^(d)For these parameters, N = 5 for Treatment C.

The percent times during which observations fall above (% TimeAbove), within (% TimeWithin), and below (% TimeBelow) the reference range are computed for each subject and are presented in Tables 14.2.1.18, 14.2.1.19 and 14.2.1.20 for Treatments A, B and C, respectively. These results are also summarized in text Table 11.4.2.3.3.

The percent of subjects with C_(avg) values for serum Estradiol above, within, and below the reference range is calculated for each treatment and are presented in Table 14.2.1.21. These results are also summarized in text Table 11.4.2.3.3.

11.4.2.4 Pharmacodynamic Analysis

No pharmacodynamic analysis is planned or performed during this study.

11.4.7 Pharmacokinetic and Statistical Conclusions

In this Phase II study, subjects are randomized into three treatment arms (4.0% TBS-1 administered t.i.d. and 4.5% TBS-1 administered bid. and t.i.d.). The treatments are administered for one week by intra-nasal route, in a parallel design. At the end of one week, the three treatments are compared by conducting a 24 hour pharmacokinetic investigation of the systemic absorption of the drug product Testosterone, and its two physiological metabolites Dihydrotestosterone and Estradiol.

Testosterone

The pharmacokinetic profile of TBS-1 following single and repeat dosing is examined in 2 previous studies (TST-PKP-01-MAT/04 and TST-DF-02-MAT/05). It is demonstrated in these studies that Testosterone is well absorbed following intra-nasal administration. The maximal serum concentration is reached after 1-2 hours post administration. In the current study, the Testosterone formulations (4.0% TBS-1 is administered t.i.d. and 4.5% TBS-1 is administered bid. and t.i.d.) are rapidly absorbed with a peak concentration reached within 36 minutes to 1 hour 6 minutes (mean T_(max)) following intra-nasal administration. The maximum Testosterone concentration over the 24-hour interval is observed during the first administration (0-10 hours) in approximately 57% to 71% of the hypogonadal men while approximately 29% to 43% of the subjects had their maximum 24-h Testosterone concentration during the subsequent administrations.

When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a greater AUC is observed following the first administration compared to the two subsequent administrations (AUC₀₋₁₀: 4178.68 and 4355.19 h*ng/dL>AUC₁₀₋₁₆: 2635.05 and 2301.51 h*ng/dL<AUC₁₆₋₂₄: 3016.52 and 2766.97 h*ng/dL for Treatments A and C, respectively). A greater AUC is observed for the second administration when compared to the first administration for Treatment B (AUC₀₋₁₀: 4451.64 h*ng/dL˜AUC₁₀₋₂₄: 5264.19 h*ng/dL). The difference in AUC between administrations for both the t.i.d. and b.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-τ) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-τ): 9920.07, 9781.39 and 9505.03 h*ng/dL for Treatments A, B and C, respectively).

Although the mean C_(max) is similar between Treatments A and C, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 786 and 857 ng/dL>C_(max 10-16): 698 and 675 ng/dL>C_(max 16-24): 556 and 595 ng/dL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 894 ng/dL˜C_(max 10-24): 846 ng/dL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods that are elapsed between each administration. The mean C_(max) calculated over the 24-hour dosing interval, is slightly greater for Treatment B (150 μL of 4.5% gel (b.i.d.)) (C_(max): 1050 ng/dL) comparatively to Treatments A and C (C_(max): 830 and 883 ng/dL, respectively). The upper limit of the physiological reference range (1050 ng/dL) is exceeded by 1 of 8 subjects for Treatment A and 3 of 7 subjects for Treatments B and C.

A trend toward a slight decrease in C_(avg) is observed when administrations are compared separately for t.i.d. and b.i.d. treatments (C_(avg 0-10): 418 and 436 ng/dL>C_(avg 10-16): 439 and 384 ng/dL>C_(avg 16-24): 377 and 346 ng/dL for Treatments A and C, respectively and C_(avg 0-10): 445 ng/dL>C_(avg 10-24): 376 ng/dL for Treatment B). The difference in C_(avg) between administrations could be due to the different time periods that are elapsed between each administration. The mean C_(avg) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 413, 408, 396 ng/dL for Treatments A, B and C, respectively).

These results suggest a decrease in exposure (AUC, C_(avg) and C_(max)) between each dose for the t.i.d. administrations (Treatments A and C), but not for the b.i.d. administration (Treatment B). This decrease in exposure for the t.i.d. administrations could be partly explained by the negative feedback on endogenous Testosterone production from the HPG axis. In other words, due to the smaller time intervals between each administration for the t.i.d. groups, the recovery of the HPG system from negative feedback would be less that for the b.i.d. group.

Independently of the formulation, approximately 86%-88% of the subjects had an average drug concentration (C_(avg)) within the physiological reference range (300 to 1050 ng/dL), 13%-14% of the subjects had a C_(avg) below the reference range and no subjects had a C_(avg) above the reference range.

The period of time during a day (24 hours) for which serum Testosterone concentrations are below, within and above the physiological reference range is covered respectively 30 to 35%, 59% to 68% and 0% of the 24-hour period for all formulations. That is to say that the testosterone levels are within normal range for about 14 to 16 hours a day.

Dihydrotestosterone

The Dihydrotestosterone peak concentration is reached within 1 hour 24 minutes and 2 hours 23 minutes (mean T_(max)) following the TBS-1 administrations. When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a trend toward a decrease in AUC with subsequent administrations is observed (AUC₀₋₁₀: 345.77 and 411.10 h*ng/dL>AUC₁₀₋₁₆: 186.33 and 222.62 h*ng/dL>AUC₁₆₋₂₄: 269.16 and 275.21 h*ng/dL for Treatments A and C, respectively). Comparable AUC is observed for both administrations of Treatment B (AUC₀₋₁₀: 402.77 h*ng/dL˜AUC₁₀₋₂₄: 543.29 h*ng/dL). The difference in AUC between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-τ) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-τ): 818.95, 946.89 and 909.68 h*ng/dL for Treatments A, B and C, respectively).

Although the mean C_(max) is similar between the t.i.d. formulations, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 51.4 and 59.0 ng/dL>C_(max 10-16): 44.2 and 48.9 ng/dL>C_(max 16-24): 41.3 and 42.6 ng/dL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 56.8 ng/dL˜C_(max 10-24): 54.6 ng/dL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean C_(max) is calculated over the 24-hour dosing interval, is comparable for all treatments (C_(max): 52.2, 61.0 and 60.3 ng/dL for Treatments A, B and C, respectively). The upper limit of the physiological reference range (97.8 ng/dL) is not exceeded by any subjects for any treatment.

The C_(avg) calculated by administration are comparable between treatments and administrations (C_(avg 0-10): 34.6 and 41.1 ng/dL>C_(avg 10-16): 31.1 and 37.1 ng/dL>C_(avg 16-24): 33.6 and 34.4 ng/dL for Treatments A and C, respectively and C_(avg 0-10): 40.3 ng/dL>C_(avg 10-24): 38.8 ng/dL for Treatment B). The mean C_(avg) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 34.1, 39.5, 37.9 ng/dL for Treatments A, B and C, respectively).

Approximately 63% of subjects had their C_(avg) included in the physiological reference range for DHT (25.5 to 97.8 ng/dL) following administration of Treatment A, whereas this number rises to about 86% when Treatments B and C are administered. No subject had their C_(avg) above the normal range while 38% and 14% of the subjects have their C_(avg) below the normal range for Treatment A and both Treatments B and C, respectively.

The period of time during a day (24 hours) for which serum DHT concentrations are below, within and above the physiological reference range is covered respectively 32.64%, 67.36% and 0% for Treatment A, 26.22%, 73.78% and 0% for Treatment B and 13.87%, 86.13% and 0% for Treatment C. That is to say that the DHT levels are within normal range for about 16, 18 and 21 hours a day for Treatments A, B and C, respectively.

Estradiol

The Estradiol peak concentration is reached within 1 hour 12 minutes and 2 hours 41 minutes (mean T_(max)) following the TBS-1 administrations.

When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a trend toward a decrease in AUC with subsequent administrations is observed (AUC₀₋₁₀: 234.96 and 267.78 h*pg/mL>AUC₁₀₋₁₆: 144.76 and 144.30 h*pg/mL<AUC₁₆₋₂₄: 153.02 and 177.97 h*pg/mL for

Treatments A and C, respectively). Comparable AUC is observed for both administrations of Treatment B (AUC₀₋₁₀: 242.02 h*pg/mL˜AUC₁₀₋₂₄: 295.12 h*pg/mL). The difference in AUC between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-τ) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-τ): 530.27, 537.16 and 601.91 h*pg/mL for Treatments A, B and C, respectively).

Although the mean C_(max) is similar between the t.i.d. formulations, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 36.8 and 35.5 pg/mL>C_(max 10-16): 28.9 and 31.5 pg/mL>C_(max 16-24): 27.2 and 26.9 pg/mL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 35.8 pg/mL˜C_(max 10-24): 30.6 pg/mL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean C_(max) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(max): 37.9, 36.2 and 36.4 pg/mL for Treatments A, B and C, respectively). The upper limit of the physiological reference range (81 pg/mL) is not exceeded by any subjects for any treatment.

e C_(avg) calculated by administration are comparable between treatments and administrations (C_(avg 0-10): 23.5 and 26.8 pg/mL>C_(avg 10-16): 24.1 and 24.0 pg/mL>C_(avg 16-24): 19.1 and 22.2 pg/mL for Treatments A and C, respectively and C_(avg 0-10): 24.2 pg/mL>C_(avg 10-24): 21.1 pg/mL for Treatment B). The mean C_(avg) is calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 22.1, 22.4, 25.1 pg/mL for Treatments A, B and C, respectively).

All subjects have their C_(avg) included in the physiological reference range for E₂ (3 to 81 pg/mL) following administration of all treatments. All subjects have E₂ concentrations within the normal range over the 24 hours period. No subjects have E₂ levels below or above the normal range at any time of the day.

12. Safety Evaluation 12.1 Extent of Exposure

Subjects use the drug for 7 days at two sites and 8 days in another.

12.2 Adverse Events

12.2.1 Brief Summary of Adverse Events

There are eight adverse events that occurred in six subjects. Six of the events occur during treatment A and two occur during treatment B. Subjects 01-002 and 01-007 both experience dizziness and both are indicated as possibly related to the study drug. Subject 01-002 has moderate severity which resolved after 5 days. Seven of the 8 adverse events are mild. Six of the 8 events are not related to study drug. Individual 02-004 is classified as having anemia by the investigator. The hemoglobin is at the minimal normal level and is deemed unrelated to the drug. Table 12.2.2 summarizes the events.

12.2.2 Display of Adverse Events

TABLE 12.2.2 Adverse Events Preferred Relation to Duration Treatment Subject Age Term Severity Drug Start Date End Date (days) A 01-002 40 Dizziness MODERATE POSSIBLY 2010 Oct. 25 2010 Oct. 30 5 RELATED A 01-007 49 Dizziness MILD POSSIBLY 2010 Oct. 23 2010 Oct. 28 5 RELATED A 02-004 53 Anemia MILD NOT 2010 Oct. 4 RELATED A 03-006 73 Pain of skin MILD NOT 2010 Sep. 27 2010 Nov. 4 37 RELATED A 03-006 73 Excoriation MILD NOT 2010 Sep. 2 2010 Nov. 4 62 RELATED A 03-006 73 Excoriation MILD NOT 2010 Sep. 27 2010 Nov. 4 37 RELATED B 03-001 59 Respiratory MILD NOT 2010 Sep. 5 2010 Sep. 13 8 tract RELATED congestion B 03-005 62 Gastrooesopha- MILD NOT 2010 Sep. 14 2010 Sep. 27 13 geal reflux RELATED disease

2.2.4 Listing of Adverse Events by Subjects

Table 12.2.2 list of adverse events by subject.

12.3 Deaths, Other Serious Adverse Events, and Other Significant Adverse Events

There are no deaths, other serious adverse events or other significant adverse events during the course of this study.

12.4.2 Evaluation of Each Laboratory Parameter

There are no clinically significant changes in laboratory values from the beginning to the end of the study as determined by the principle investigators. All subjects did have some abnormal values at the initial visit and/or at the third visit. There are no consistent changes throughout the visits.

Subject 01-007 had a uric acid level of 539 U/L with 289 as the upper end of normal at the third visit. There are elevated glucose values in about half the subjects compared to a normal first visit value. This is spread across all three dosages and are only slightly elevated. There is no clinical significance.

12.5 Vital Signs, Physical Findings, and Other Observations Related to Safety

There are no meaningful or significant changes in vital signs after test drug administration.

12.6 Safety Conclusions

The TBS-1 gel demonstrates in this and other studies that it is safe for use. There are no serious adverse events or any events of consequence during this PK study or during the seven days of self administration. Tables 14.3.2.1 through 14.3.2.8 show all the laboratory values for visit 1 and visit 3.

13. Discussion and Overall Conclusions

The primary objective of this study is to determine the bioavailability of a 4.0% TBS-1 gel (applied t.i.d.) and 4.5% TBS-1 gel (applied b.i.d. and t.i.d.) in hypogonadal men.

In a previous study, Nasobol-01-2009, a 3.2% Testosterone gel is used to deliver 4.0 mg, 5.5 mg and 7.0 mg of Testosterone intra-nasally using gel volumes of 125 μL, 172 μL and 219 μL, respectively. In this study, 5.0 mg, 5.65 mg and 6.75 mg of Testosterone are administered in gel volumes of 125 μL, 125 μL, and 150 μL, respectively. This study allowed investigating the delivery of similar Testosterone amounts in much smaller volumes.

The secondary objective of this study is to establish a safety profile for TBS-1. In this Phase II study, subjects are randomized into three treatment arms (4.0% TBS-1 administered t.i.d. and 4.5% TBS-1 administered bid. and t.i.d.). The treatments are administered for one week by intra-nasal route, in a parallel design. At the end of one week, the three treatments are compared by conducting a 24 hour pharmacokinetic investigation of the systemic absorption of the drug product Testosterone, and its two physiological metabolites Dihydrotestosterone and Estradiol.

There are eight adverse events described by six subjects. Six of the events occurred during treatment A and two occurred during treatment B. Subjects 01-002 and 01-007 both experienced dizziness and both are indicated as possibly related to the study drug. The remainder are unrelated to study drug.

There are no vital signs or laboratory changes that are significant or meaningful. No erythrocytosis, anemia or infections are observed after measurement of complete blood counts at screening and close-out. Clinical chemistry and urinalysis showed no changes at close-out in hypo or hyperglycemia, renal function, liver function, skeletal/heart muscle damage or changes in calcium homeostasis. There are no clinically significant changes to the nasal mucosa.

The PK population is defined as subjects who received the Treatment A, B or C, and who completed the study without major protocol violation or for whom the PK profile can be adequately characterized. The PK population is used for the analysis of PK data. Based on these criteria, twenty-two (22) subjects are included in the PK population.

Testosterone

The pharmacokinetic profile of TBS-1 following single and repeat dosing is examined in 2 previous studies (TST-PKP-01-MAT/04 and TST-DF-02-MAT/05). It is demonstrated in these studies that Testosterone is well absorbed following intra-nasal administration. The maximal serum concentration is reached after 1-2 hours post administration. In the current study, the Testosterone formulations (4.0% TBS-1 administered t.i.d. and 4.5% TBS-1 administered bid. and t.i.d.) are rapidly absorbed with a peak concentration reached within 36 minutes to 1 hour 6 minutes (mean T_(max)) following intra-nasal administration. The maximum Testosterone concentration over the 24-hour interval is observed during the first administration (0-10 hours) in approximately 57% to 71% of the hypogonadal men while approximately 29% to 43% of the subjects had their maximum 24-h Testosterone concentration during the subsequent administrations.

When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a greater AUC is observed following the first administration compared to the two subsequent administrations (AUC₀₋₁₀: 4178.68 and 4355.19 h*ng/dL>AUC₁₀₋₁₆: 2635.05 and 2301.51 h*ng/dL<AUC₁₆₋₂₄: 3016.52 and 2766.97 h*ng/dL for Treatments A and C, respectively). A greater AUC is observed for the second administration when compared to the first administration for Treatment B (AUC₀₋₁₀: 4451.64 h*ng/dL˜AUC₁₀₋₂₄: 5264.19 h*ng/dL). The difference in AUC between administrations for both the t.i.d. and b.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-t) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-t): 9920.07, 9781.39 and 9505.03 h*ng/dL for Treatments A, B and C, respectively).

When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean C_(max) is similar between formulations, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 786 and 857 ng/dL>C_(max 10-16): 698 and 675 ng/dL>C_(max 16-24): 556 and 595 ng/dL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 894 ng/dL˜C_(max 10-24): 846 ng/dL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean C_(max) calculated over the 24-hour dosing interval, is slightly greater for Treatment B (150 μL of 4.5% gel (b.i.d.)) (C_(max): 1050 ng/dL) comparatively to Treatments A and C (C_(max): 830 and 883 ng/dL, respectively). The upper limit of the physiological reference range (1050 ng/dL) is exceeded by 1 of 8 subjects for Treatment A and 3 of 7 subjects for Treatments B and C.

A trend toward a slight decrease in C_(avg) is observed when administrations are compared separately for t.i.d. and b.i.d. treatments (C_(avg 0-10): 418 and 436 ng/dL>C_(avg 10-16): 439 and 384 ng/dL>C_(avg 16-24): 377 and 346 ng/dL for Treatments A and C, respectively and C_(avg 0-10): 445 ng/dL>C_(avg 10-24): 376 ng/dL for Treatment B). The difference in C_(avg) between administrations could be due to the different time periods elapsed between each administration. The mean C_(avg) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 413, 408, 396 ng/dL for Treatments A, B and C, respectively).

These results suggest a decrease in exposure (AUC, C_(avg) and C_(max)) between each dose for the t.i.d. administrations (Treatments A and C), but not for the b.i.d. administration (Treatment B). This decrease in exposure for the t.i.d. administrations could be partly explained by the negative feedback on endogenous Testosterone production from the HPG axis. In other words, due to the smaller time intervals between each administration for the t.i.d. groups, the recovery of the HPG system from negative feedback would be less that for the b.i.d. group.

Independently of the formulation, approximately 86%-88% of the subjects had an average drug concentration (C_(avg)) within the physiological reference range (300 to 1050 ng/dL), 13%-14% of the subjects had a C_(avg) below the reference range and no subjects had a C_(avg) above the reference range.

The period of time during a day (24 hours) for which serum Testosterone concentrations are below, within and above the physiological reference range covered respectively 30 to 35%, 59% to 68% and 0% of the 24-hour period for all formulations. That is to say that the Testosterone levels are within normal range for about 14 to 16 hours a day.

Dihydrotestosterone

The Dihydrotestosterone peak concentration is reached within 1 hour 24 minutes and 2 hours 23 minutes (mean T_(max)) following the TBS-1 administrations. When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a trend toward a decrease in AUC with subsequent administrations is observed (AUC₀₋₁₀: 345.77 and 411.10 h*ng/dL>AUC₁₀₋₁₆: 186.33 and 222.62 h*ng/dL>AUC₁₆₋₂₄: 269.16 and 275.21 h*ng/dL for Treatments A and C, respectively). Comparable AUC is observed for both administrations of Treatment B (AUC₀₋₁₀: 402.77 h*ng/dL˜AUC₁₀₋₂₄: 543.29 h*ng/dL). The difference in AUC between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-t) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-t): 818.95, 946.89 and 909.68 h*ng/dL for Treatments A, B and C, respectively).

Although the mean C_(max) is similar between the t.i.d. formulations, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 51.4 and 59.0 ng/dL>C_(max 10-16): 44.2 and 48.9 ng/dL>C_(max 16-24): 41.3 and 42.6 ng/dL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 56.8 ng/dL˜C_(max 10-24): 54.6 ng/dL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean C_(max) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(max): 52.2, 61.0 and 60.3 ng/dL for Treatments A, B and C, respectively). The upper limit of the physiological reference range (97.8 ng/dL) is not exceeded by any subjects for any treatment.

The C_(avg) calculated by administration are comparable between treatments and administrations (C_(avg 0-10): 34.6 and 41.1 ng/dL>C_(avg 10-16): 31.1 and 37.1 ng/dL>C_(avg 16-24): 33.6 and 34.4 ng/dL for Treatments A and C, respectively and C_(avg 0-10): 40.3 ng/dL>C_(avg 10-24): 38.8 ng/dL for Treatment B). The mean C_(avg) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 34.1, 39.5, 37.9 ng/dL for Treatments A, B and C, respectively).

Approximately 63% of subjects had their C_(avg) included in the physiological reference range for DHT (25.5 to 97.8 ng/dL) following administration of Treatment A, whereas this number rises to about 86% when Treatments B and C are administered. No subject had their C_(avg) above the normal range while 38% and 14% of the subjects had their C_(avg) below the normal range for Treatment A and both Treatments B and C, respectively.

The period of time during a day (24 hours) for which serum DHT concentrations are below, within and above the physiological reference range covered respectively 32.64%, 67.36% and 0% for Treatment A, 26.22%, 73.78% and 0% for Treatment B and 13.87%, 86.13% and 0% for Treatment C. That is to say that the DHT levels are within normal range for about 16, 18 and 21 hours a day for Treatments A, B and C, respectively.

Estradiol

The Estradiol peak concentration is reached within 1 hour 12 minutes and 2 hours 41 minutes (mean T_(max)) following the TBS-1 administrations.

When TBS-1 administrations are compared separately for the t.i.d. treatments, although the mean AUC is similar between formulations, a trend toward a decrease in

AUC with subsequent administrations is observed (AUC₀₋₁₀: 234.96 and 267.78 h*pg/mL>AUC₁₀₋₁₆: 144.76 and 144.30 h*pg/mL<AUC₁₆₋₂₄: 153.02 and 177.97 h*pg/mL for Treatments A and C, respectively). Comparable AUC is observed for both administrations of Treatment B (AUC₀₋₁₀: 242.02 h*pg/mL˜AUC₁₀₋₂₄: 295.12 h*pg/mL). The difference in AUC between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean AUC_(0-t) calculated over the 24-hour dosing interval, is comparable between all treatments (AUC_(0-t): 530.27, 537.16 and 601.91 h*pg/mL for Treatments A, B and C, respectively).

Although the mean C_(max) is similar between the t.i.d. formulations, a trend toward a decrease in C_(max) with subsequent administrations is observed (C_(max 0-10): 36.8 and 35.5 pg/mL>C_(max 10-16): 28.9 and 31.5 pg/mL>C_(max 16-24): 27.2 and 26.9 pg/mL for Treatments A and C, respectively). Comparable mean Testosterone C_(max) is observed for both administrations of Treatment B (C_(max 0-10): 35.8 pg/mL˜C_(max 10-24): 30.6 pg/mL). The difference in C_(max) between administrations for the t.i.d. formulations could be due to the different time periods elapsed between each administration. The mean C_(max) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(max): 37.9, 36.2 and 36.4 pg/mL for Treatments A, B and C, respectively). The upper limit of the physiological reference range (81 pg/mL) is not exceeded by any subjects for any treatment.

The C_(avg) calculated by administration are comparable between treatments and administrations (C_(avg 0-10): 23.5 and 26.8 pg/mL>C_(avg 10-16): 24.1 and 24.0 pg/mL>C_(avg 16-24): 19.1 and 22.2 pg/mL for Treatments A and C, respectively and C_(avg 0-10): 24.2 pg/mL>C_(avg 10-24): 21.1 pg/mL for Treatment B). The mean C_(avg) calculated over the 24-hour dosing interval, is comparable for all treatments (C_(avg): 22.1, 22.4, 25.1 pg/mL for Treatments A, B and C, respectively).

All subjects had their C_(avg) included in the physiological reference range for E₂ (3 to 81 pg/mL) following administration of all treatments. All subjects had E₂ concentrations within the normal range over the 24 hours period. No subjects had E₂ levels below or above the normal range at any time of the day.

Conclusions

The TBS-1 formulations (4.0% TBS-1 gel (applied t.i.d.) and 4.5% TBS-1 gel (applied b.i.d. and t.i.d.)) are rapidly absorbed with mean Testosterone peak observed within 1 hour.

Overall, the Testosterone exposure (AUC_(0-t) and C_(max)) at steady-state is comparable between all treatments.

Independently of the formulation, approximately 86%-88% of the subjects had an average Testosterone drug concentration (C_(avg)) within the physiological reference range (300 to 1050 ng/dL).

The Testosterone levels are within normal range for about 14 to 16 hours a day.

TBS-1 is safe for intranasal administration at the dosages and frequency indicated. There are no meaningful adverse events, changes in vital signs or changes in laboratory results when compared to baseline.

Based on these results, no clear evidence is found to indicate a better performance from one of the formulations.

Example 9 TBS1A Report for 4% and 8% Bulk Gel Objective:

To follow up on IMP-Clinical batch manufacture. Main points concern process flow and bulk appearance on stability.

-   -   Process flow improvement     -   Viscosity of bulk Gel     -   Stability (re-crystallization)     -   Evaluation of alternate materials sources and grades     -   In Vivo results, formulation changes to impact onset of release     -   Testing of trials using Franz Cell, trial selection

List of Raw-materials identified for use in trials:

Material name Grade Spec # Source Comments Castor Oil (Crystal O) RM004A Cas-Chem Castor Oil (Virgin) RM004B — Labrafil M1944CS RM002A Gattfosse DMI — RM009A Croda Transcutol P — RM008A Gattfosse Plasdone K17 RM011A ISP Plasdone S630 RM013A ISP Plasdone K29-32 Sample ISP Plasdone K90 Sample ISP HPC Klucel HF RM014A Hercules HPC Nisso H Sample Nisso HPC Nisso M Sample Nisso HPC Nisso L Sample Nisso Cab-O-Sil M-5P RM003A Cabot Aerosil 200 RM003B Evonik Purified water — — Trimel Testosterone micronized RM001A Proquina Oleic Acid Super- sample Croda refined Testosterone Not RM Proquina micronized

Equipment Used:

In addition to the Silverson High Shear mixer, used only during the manufacture of the TBS1A IMP Clinical batches, included also a propeller type mixing unit for the trials on several pre-mix operations. The only application for the High shear action is for dispersion of the active in the Co-Solvents.

For more uniform mixing and control of temperature, recommend a jacketed container with wiping blades to remove material from inner bowl wall (especially critical for uniform bulk temperature during heating as well as cooling cycles.

Background Info on IMP Bath Manufacture

Observation during the IMP Clinical batch manufacture included high viscosity during preparing the pre-mixture of the DMI/Transcutol co-solvent mix consisting of PVP K17/5640, Klucel HF and Testosterone micronized. Mixture resulting in a sticky mass when added to the Castor oil using the high shear mixer set up. With the same high shear mixer set up for the addition of the Cab-O-Sil (referenced in future to SiO2) could not obtain a vortex to incorporate the material and required additional manual mixing during addition stage, hence the recommendation for propeller type mixing unit). Even though the material was viscous during that addition stage, on further mixing the viscosity of the final Bulk Gel dropped to approximately 1,500-2,000 cps. Mixing time and speed had to be controlled not to overshoot targeted gel temperature (no cooling system).

Outline of Trials:

The initial trials (Placebo) concentrated on changing the order of addition to identify impact on viscosity. Previous process included the addition of the SiO2 at the final stage (see comments above), changed to dispersion of the SiO2 into the Castor oil prior to addition of the alternate active mixture. The resulting viscosity of the Castor Oil/SiO2 mixture, used various percentages, increased with the addition of a small percentage of Arlasolve (DMI).

Next step was to duplicate these results using the active mixture (Co-solvents/PVP/HPC/active) and added that mixture to the premix of Castor oil and SiO2. This however resulted in a low viscosity solution, indicating an impact of the active mixture on formation of a viscous gel.

Since the co-solvent mix without additional materials resulted in an increase of viscosity, the quantities of solvent were split into 2 parts, adding part of the solvent mix only to the Oil mixture and remaining solvent mix used to disperse the PVP, HPC and active. The active mixture with the reduced co-solvent ended up more viscose, plus similar low viscosity when added to the castor Oil premix. Additional trials included the prep of active in only DMI (no PVP) and obtained good viscosity. HPC was prepared separately in the Transcutol P, creating problems of stringing when added to the mixture (similar to IMP observations). Addition of SiO2 at a level of 0.1-0.3% resolved the problem.

The above process to dissolve active in the Co-solvents is sufficient and doesn't require PVP to increase solubility for the 4% formulation, however not sufficient co-solvents in the formulation to achieve solubility for the 8% strength. Trials on the 8% included an alternate successful approach for preparing the active dispersion containing PVP by including SiO2 into that mixture. As demonstrated on evaluation trials evaluating impact of SiO2 added to the DMI as well as Transcutol P, resulted in good viscosity forming with DMI, however not with Transcutol. Active dispersion therefore id prepared by dissolving the PVP in DMI only, followed by addition of the active at 55 C (50-60 C) and portion of available SiO2.

Please note that this process was only developed during the trial work on the 8%, hence it can be scaled down to the 4% strength if PVP indicate additional functionality (Franz Cell test).

Comments related to addition of purified water (noted in Table xxx) indicate increase in viscosity with trials containing HPC, no viscosity increase in trials using only PVP. These trials were only included for information to study water uptake and impact on viscosity after application into the nasal cavity.

Critical step during HPC set up is to provide at least 24 hours of solvating to obtain a clear solution.

As outlined in the trial objectives, formulation ratios were implemented using also alternate grades and sources of materials and are identified in the formulation table. To identify the impact of the process change (such as reaction of viscosity increase adding the co-solvents), performed trials to study impact if related to DMI or Transcutol P. Trials were initiated to disperse SiO2 (at the same ratio as used for Castor Oil mixture) in DMI only as well as in Transcutol P only. The Mixture with the DMI resulted in a viscous mixture while Transcutol P mixture was very fluid.

Similar trials were initiated to use the co-solvents individually to study solubility of the Polymers as well as active for potential reduction in Transcutol P. No noticeable difference in solubility using the mixture or individual solvents at the 4% strength. However, if PVP and HPC are prepared only in DMI, observed separation of the two materials when stored overnight (not apparent when mixed in the co-solvent mixture). To eliminate the stickiness of the dispersion when adding the active/polymer mixture, removed the HPC from the formulation and using PVP only (individual grades K17-K29/32-K90, no mixtures). This resulted in various degrees of viscosity related to the grade used.

Material also included the use of Labrafil M 1944 CS and are outlined in batch description and selected for testing in Franz Cell.

Comments:

The various trials are outlined below for 4% strength as well as 8%.

Trial lots of both strength have been selected for testing on the Franz Cell. Selected lots are identified.

All trials will be monitored for physical evidence of re-crystallization and change in appearance (separation), tested for change in viscosity. Viscosity values of the trials will be documented and updated

Pending Franz Cell result evaluation, optimization of formulation and process can be implemented. This is critical to identify since the trial outline did not include impact on viscosity related to all process parameters (need to include analytical testing and stability data).

Observations during viscosity test using the Brookfield Viscometer Model DV-II+, with Spindle #6, at 50 rpm for 30 seconds, did actually show an increase in viscosity values over the test time in samples prepared with higher viscosity grade HPC. This can be attributed to the stickiness of the Gel causing agglomeration to the spindle shaft and disk creating a drag (not a true viscosity value of the results reported). The bulk Gel of several trials is not thixotropic. Also tested on some trials viscosity at 37 C.

Tested several trials using the new Haupt method with spindle 4 at 6 rpm.

The various attached tables show the trial numbers for active Gels, pre-mixes and Placebos

Discussion and Considerations for Follow Up Trials with Both Strength

Even though ‘viscosity improvement’ was not the primary target to initiate trials, it was certainly a designed effort to study the cause for low viscosity considering the high percentage of SiO2 present in the formulation. A cross check against SiO2 alternate source comparison did not indicate major differences, nor did various ratios of Co-Solvents, limited adjustment since a certain percentage required to dissolve the Testosterone. Changes in grades of PVP indicated impact on viscosity when used in the active dispersion, however not when added to the rest of the mixture. Changes in grades of HPC (used alternate source of fine material) showed impact on the final Gel, however the higher the Molecular weight of the HPC, impact of stickiness and stringing in the final Gel. Testing viscosity after several weeks did show a separation in the Gel of viscose settlement on the bottom of the container.

With indication of SiO2 retaining Testosterone, adding more to increase viscosity was not an option, aim was to reduce the % used. especially for the TBS1A 4% strength which indicated a much higher percentage of T retained compared to the 8% TBS1A. Target was to at least obtain the same ratio of SiO2 to T of the 8% strength for the 4% strength (hence aimed for scale down to 3%). With the trials completed and showing impact on viscosity related to process and formulation changes, a reduction in SiO2 for the definitely possible for the 4% strength that would also include the use of PVP in the formulation by taking advantage of the process change on the 8% strength. The above is only based on viscosity; however impact on the changes in formulation to slow down initial absorption rate in vivo can only be evaluated from the data obtained on the trials used for the analytical test using the Franz Cell. These results will be reviewed and evaluated with potential recommendations for further trials to either duplicate earlier trials or based on DOE.

The attached Tables for viscosity show the date of manufacture and latest test results (to help with trial selection on Franz Cell). In the Comment column original data will be reference or referenced in the Trial process description.

Further alternate material source evaluation is recommended once a primary formulation and process for each strength has been established for direct comparison.

Formulation/Composition of TBS1A—4%

TABLE 1A (See the formulations in the Examples above and including Example 10) SiO2 % Trial Active Castor oil Labrafil PVP grade DMI TranscutolP HPC Nisso C = Cabosil number % % % % % % % A = Aerosil200 RD11037 4 52  000000 K17 = 3   25    10  0000000 C = 4   S630 = 2    RD11038 4 57  000000 K17 = 3   20    10  0000000 C = 4   S630 = 2    RD11039 4 29    29 K17 = 3   20    10  0000000 C = 3   S630 = 2    RD11040 4 57 0000000  0000000 25    10 00000000 C = 4   6 + 4 RD11041 4 53 0000000 K17 = 3   25    10  0000000 C = 3   S630 = 2    6 + 4 RD11042 4 29    29 00000000 25    10  000000 C = 3   6 + 4 (split) RD11050 4 66.7  000000 K17 = 3   24  0000000 N − H = 0.3  A = 2   20 + 4 RD11050A 4 66.7  000000 K17 = 3   24  0000000 N − H = 0.3  1% additional 20 + 4 to final 11050 RD11051 4 66.7  000000 K30 = 3   24  0000000 N − M = 0.3   A = 2   20 + 4 RD11051A 4 66.7  000000 K30 = 3   24  0000000 N − M = 0.3   1% additional 20 + 4 to final 11051 RD11053 4 61.7  000000 K17 = 3   22     6 N − H = 0.3  A = 3   16 + 6 4 + 2 RD11054 4 61.4  000000 K30 = 3   23     5 N − M = 0.6   A = 3   16 + 7 4 + 1 RD11055 4 62.0  000000 K90 = 3   23     5  0000000 C = 3   16 + 7 4 + 1 RD11056 4 62.0  000000 K90 = 3   28   00000  0000000 C = 3   20 + 8 RD11059 4 75.0  000000 K30 = 2.5 14     2  0000000 C = 2.5 10 + 4 RD11060 4 71.5  000000 K30 = 2.0 18     1 00000000 C = 3.5  9 + 9 RD11061 4 71.0    2 K17 = 2   16     2  0000000 C = 3   RD11062 4 62.35 0000000 K17 = 1.5 22     6 N − H = 0.15 A = 3   K30 = 1.0  6 + 16 2 + 4 RD11063 4 70.5 00000oo K17 = 1.5 18 00000000 N − H = 0.2  A = 4   K30 = 1.5  6 + 12 RD11064 Transfer Add 0.3% Increase Formula from H2O in includes RD11062 viscosity HPC RD11065 Transfer Add 0.3% Increase Formula from H2O in includes RD11063 viscosity HPC RD11066 Transfer Add 0.3% No N0 HPC from H2O increase RD11041 in viscosity RD11070 Transfer Add 0.3% No N0 HPC from H2O increase RD11037 in viscosity RD11071 Transfer Add 0.3% No N0 HPC from H2O increase RD11042 in viscosity RD11072 Transfer Add 0.3% No N0 HPC from H2O increase RD11040 in viscosity RD11073 4 70.5  000000  0000000 16     6 (3) N − M = 0.5  A = 3    10 + 6 (3) (0.25) RD11074 Transfer Add 0.3% Transfer Add 0.3% H2O from H2O from RD11073 RD11040 RD11075 4 68.0  000000 K30 = 1.0 16  0000000 See HPC A = 3   (base)  6 + 10 pre-mixes RD11076 Base of — — — — — Addition — RD11075 RD11067 RD11077 Base of — — — — — Addition — RD11075 RD11068 RD11078 Base of — — — — — Addition — RD11075 RD11069 RD11079 Transfer Add 0.3% — — — — Formula — from H2O includes RD11076 HPC RD11080 Transfer Add 0.3% — — — — Formula — from H2O includes RD11077 HPC RD11081 Transfer Add 0.3% — — — — Formula — from H2O includes RD11078 HPC RD11082 4 81.0  000000  0000000 10 See See 00000000 See RD11073 (3 RD11073 RD11073 (3 (0.25) RD11085 4 70.7  000000  0000000 16     6 N − L = 0.2  A = 2.8 10 + 6 N − M = 0.3   RD11086 4 70.7  000000  0000000 16     6 N − L = 0.2  A = 2.8 Add 0.3% 10 + 6 N − M = 0.3   H2O

Lot # RD11037

Process duplication of IMP batch (4%) without HPC. K17 and S630 dissolved in DMI/Transcutol mixture followed by addition of the active. Clear solution. Castor oil preheated and added the above active mixture. Clear solution observed. Followed with the addition of the Cabosil with low shear. Viscosity at time of manufacture 500 cps, followed with test after 48 hours resulted in 620 cps.

Lower viscosity primarily due to missing HPC (note that IMP 4% had approx 1,500 cps)

Lot # RD11038

Change in order of addition using the same formulation with a reduction of DMI/Transcutol and adjusted with castor oil. Cabosil was mixed into the Castor oil obtaining a clear viscous solution. The active mixture was prepared as per RD11037. Viscosity of the Castor oil/Cabosil mixture changed to 1180 cps (expected higher viscosity based on addition of Co Solvents during the Placebo trials). Potential impact of PVP and active to solvent mixture.

Lot # RD11039

Duplicated performance based on Placebo mixture also containing Labrafil in castor oil plus Cabosil (for IP). Same reaction of reduced viscosity when adding the active mixture.

Lot # RD11040

Duplicated Placebo process adding to the Castor oil/Cabosil mixture a portion of the DMI/Transcutol P co-solvent mixture. Viscosity of the oil mixture increased. Prepared the active mixture with the remaining co-solvents without the PVP and added to the oil mixture. Final viscosity of the bulk Gel was 10,400 cps. Potential for F/C.

Lot # RD11041

Process was repeated as per RD11040 including the PVP K17 and S630 with the active mixture and viscosity was reduced to 500 cps (increased to 1,500 cps after 3 weeks). Clear indication of PVP impact on lowering viscosity using K17 and S630.

Lot # RD11042

Repeat of trial with Castor oil/Labrafil addition as per RD11037, and reduced Cabosil, with active co solvent mixture but no PVP. Viscosity of 1,750 cps

The following trials were designed to identify impact of changing to higher PVP grades as well as alternate source of HPC (2 grades). Pre mixture were made as outlined in table 3 concentrating on mixtures without Labrafil, using Castor oil native and Aerosil 200.

Lot # RD11050

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI (4%). The preparation of the active mixture use the pre-mix of RD11047A (PVP K17-3%) in DMI only, added 0.3% of HPC Nisso H followed by addition of active. Active mixture was added to the Pre-mix I

Lot # RD11050A

Same basic formulation as RD11050 with change of adding to a portion additional 1% of Aerosil 200

Lot # RD11051

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI (4%). The preparation of the active mixture use the pre-mix of RD11047B (PVP K30-3%) in DMI only, added 0.3% of HPC Nisso M followed by addition of active. Active mixture was added to the Pre-mix I

Lot # RD11051A

Same basic formulation as RD11051 with change of adding to a portion additional 1% of Aerosil 200

Lot # RD11053

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI and Transcutol P. The preparation of the active mixture use the pre-mix of RD11048A (PVP K17-3%), added 0.3% of HPC Nisso H followed by addition of active. Active mixture was added to the Pre-mix I

Lot # RD11054

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI and Transcutol P. The preparation of the active mixture use the pre-mix of RD11048B (PVP K30-3%), added 0.3% of HPC Nisso H followed by addition of active. Active mixture was added to the Pre-mix I

Lot # RD11055

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI and Transcutol P. The preparation of the active mixture use the pre-mix of RD11048C (PVP K90-3%). No HPC added. Active mixture was added to the Pre-mix I

Lot # RD11056

Dispersion (pre-mix I) of Castor Oil and Aerosil 200 was prepared and viscosity increased by adding part of the DMI. The preparation of the active mixture use the pre-mix of RD11047C (PVP K90-3%). No HPC added Active mixture was added to the Pre-mix I

Lot # RD11059

Prepared mixture of Castor Oil and Cabosil (2.5%). Active was dissolved in DMI and Transcutol P. Resulted in milky appearance. Adding that mix to the Castor Oil pre-mix, mixture did not clear up. Prepared the PVP (K30) solution with DMI, added to the mix, no change in appearance however reduced viscosity.

Note, no change in evaluation adding a mixture of 0.1% HPC to appearance, slight increase in viscosity. Trial not reported under trial a lot number.

Lot # RD11060

Prepared the Castor Oil adding 3.5% Cabosil, followed by addition of a mixture of DMI/Transcutol P for thickening. The active dispersion was prepared in a PVP (K30) with DMI as co-solvent. (no HPC)

Lot # RD11061

Prepared the Castor Oil adding 3% Cabosil, followed by addition of Labrafil (2%) for thickening. The active dispersion was prepared in a DMI mixture containing PVP K17 (2%). Mix resulted in low viscosity, however could be considered for F/C test.

Lot # RD11062

Castor Oil native mixed with Aerosil 200 (3%) and added a mixture of DMI/Transcutol P (6+2) for thickening. A PVP mixture of K17 and K30 was dissolved in DMI/Transcutol P and followed with HPC H and solvate for 4 days. Mixture was reheated prior to addition of active. Castor Oil premix was heated prior to adding the active dispersion. Recommended for F/C

Lot # RD11063

Castor Oil native mixed with Aerosil 200 (4%) and added the DMI (6%) resulting in a high viscose mix. A mixture of PVP K17 and L29/32 was dissolved in DMI, plus HPC Nisso H (0.2). On overnight setup, noticed a separation, required re-mixing. Active was added to the high viscosity Castor Oil premix. To be followed up with modification to composition Potential for F/C or to use RD11065

Lot # RD11064

Addition of 0.3% to portion of lot RD11062

Lot # RD11065

Addition of 0.3% to portion of lot RD11063

Lot # RD11066

Addition of 0.3% to portion of lot RD11041

Lot # RD11070

Addition of 0.3% to portion of lot RD11037

Lot # RD11071

Addition of 0.3% to portion of lot RD11042

Lot # RD11072

Addition of 0.3% to portion of lot RD11040

Lot # RD11073

Prepared Castor Oil/Aerosil 200 pre-mixture. Dissolve in DMI (6%) without PVP, the Testosterone and add to the Castor oil pre-mix. Obtained a viscosity of 6,300 cps. In a mixture of Transcutol P and DMI disperse the HPC M (only used 0.25% of prep) and add to main mix. Proposed for F/C

Lot # RD11074

Addition of 0.3% to portion of lot RD11072

Lot # RD11075

Prepared a stock mixture to complete 3×500 g trials consisting of Castor-Oil (68%) Aerosil 200 (3%) DMI (6%). To this mix was added PVP K29-32 (1%) in DMI (10) and active. Bulk split into 3 parts to be completed for 3 trials containing different mixtures and grades of HPC Nisso in Transcutol (ref lots RD11067/68/69)

Lot # RD11076

Used bulk from RD11075 and added HPC mix RD11067 (Transcutol P with Nisso H (0.15%)

Lot # RD11077

Used bulk from RD11075 and added HPC mix RD11068 (Transcutol P with Nisso H (0.2%)

Lot # RD11078

Used bulk from RD11075 and added HPC mix RD11069 (Transcutol P with Nisso H (0.1) and M (0.1)

Lot # RD11079

Addition of 0.3% to portion of lot RD11076

Lot # RD11080

Addition of 0.3% to portion of lot RD11077

Lot # RD11081

Addition of 0.3% to portion of lot RD11078

Lot # RD11082

Trial attempt to prepare a batch without the use of SiO2 failed

Lot # RD11085

Prepared Castor-Oil pre-mix adding 2.5% Aerosil 200 followed with a mix of DMI (10) and Testosterone. Obtained viscosity of 3,100 cps. Followed with the addition of HPC Nisso L (0.2%) and Nisso M (0.3%) mixed in DMI and Transcutol plus 0.3% Aerosil 200 to reduce stickiness. Material was added without any stringing to the main mixture and obtained a viscosity of 4,800 cps at day of manufacture and 4,900 cps 3 weeks later. Proposed for F/C

Lot # RD11086

Addition of 0.3% to portion of lot RD11085

TABLE 2 TBS1A 4% strength Viscosity values using spindle 6, 20 rpm, Repeat test ref to Franz Cell: F/C Trial Manuf Lot number date Test date and values Comments RD11037 Jul. 15, 2011 Oct. 4, 2011 Clear solution, previous results in July 620 cps   940 cps and follow up test Sep. 15, 2011 was 900 cps RD11038 Jul. 15, 2011 Oct. 4, 2011 Clear solution, original test 1,180 cps, follow up 1,800 cps Sep. 15, 2011 1,660 cps RD11039 Jul. 20, 2011 Oct. 4, 2011 Clear solution, previous results in July 980 cps 1,380 cps and follow up test Sep. 15, 2011 was 1,300 cps RD11040 Jul. 20, 2011 Oct. 4, 2011 Clear Gel, previous results in July 10,400 cps 11,040 cps  and follow up test Sep. 15, 2011 was 10,140 cps RD11041 Jul. 21, 2011 Oct. 4, 2011 Clear solution, previous results in July 500 cps 1,420 cps and follow up test Sep. 15, 2011 was 1,500 cps RD11042 Jul. 21, 2011 Oct. 4, 2011 Clear solution, test Sep. 15, 2011 was 1,720 cps 1,430 cps RD11050 Aug. 9, 2011 Oct. 4, 2011 Original comment sticky mixture, Sep. 15, 2011 Test not valid results 2,460 Do not use trial lot for F/C Poor mixture, HPC settled to bottom as a slug RD11050A Aug. 9, 2011 Oct. 4, 2011 Original comment sticky mixture, results Test not valid Sep. 15, 2011 3,000 cps (increased during test from 2,400) Do not use trial lot for F/C Poor mixture, HPC settled to bottom as a slug RD11051 Aug. 9, 2011 Oct. 4, 2011 Clear, results Sep. 15, 2011 1,940 cps  2,100 cps▴ Note: viscosity values increase during 30 sec test RD11051A Aug. 9, 2011 Oct. 4, 2011 Clear, results Sep. 15, 2011 2,560 cps  2,540 cps▴ Note: viscosity values increase during 30 sec test RD11053 Aug. 10, 2011 Oct. 4, 2011 Clear but sticky with air bubbles, results  4,500 cps▴ Sep. 15, 2011 4,060 cps Note: viscosity values increase during 30 sec test RD11054 Aug. 10, 2011 Oct. 4, 2011 Sep. 15, 2011 test HPC globules, 15,000 cps 14,000 cps▴ Do not use trial lot for F/C, Note: viscosity values increase during 30 sec test Build up of HPC on spindle RD11055 Aug. 10, 2011 Oct. 4, 2011 Sep. 15, 2011, EEEEEE EEEEEE Do not use trial lot for F/C Note, error message indicates above 20,000 tester limit at that setting RD11056 Aug. 10, 2011 Oct. 4, 2011 Sep. 15, 2011, EEEEEE EEEEEE Do not use trial lot for F/C Note, error message indicates above 20,000 tester limit at that setting RD11059 Aug. 22, 2011 Oct. 4, 2011 Do not use trial lot for F/C Test not valid Separation of HPC (?)Build up of HPC on spindle RD11060 Aug. 23, 2011 Oct. 5, 2011 Uniform texture 3,540 cps RD11061 Aug. 23, 2011 Oct. 5, 2011 Uniform texture   960 cps RD11062 Aug. 24, 2011 Oct. 5, 2011 Original viscosity 2,400 cps 3,200 cps RD11063 Aug. 24, 2011 Oct. 5, 2011 Original viscosity 1,600 cps 3,460 cps RD11064 Aug. 31, 2011 Oct. 5, 2011 Original viscosity 5,800 cps 6,440 cps Clear, thick, RD11065 Aug. 31, 2011 Oct. 5, 2011 Added .3% H2O to RD11063 Sep. 31, 2011 12,500 cps  resulted in 9,100 cps Air bubbles RD11066 Aug. 31, 2011 Oct. 5, 2011 Added .3% H2O to RD11041 Sep. 31, 2011 2,600 cps resulted in 1,500 cps Clear, thick RD11070 Aug. 31, 2011 Oct. 5, 2011 Added .3% H2O to RD110370 Sep. 31, 2011 1,540 cps resulted in 720 cps Liquid and clear RD11071 Aug. 31, 2011 Oct. 5, 2011 Added .3% H2O to RD11042 1,820 cps Sep. 31, 2011 resulted in 1,760 cps Liquid and clear RD11072 Aug. 31, 2011 Oct. 5, 2011 Added .3% H2O to RD11040 resulted in 7,920 7,920 cps cps Clear and thick, no change in viscosity RD11073 Sep. 7, 2011 Oct. 5, 2011 Started off in Sept with viscosity of 5,500 cps 9,980 cps RD11074 Sep. 7, 2011 Oct. 5, 2011 Added .3% H2O to RD11073 increases viscosity 10,100 cps  to 7,200 cps. RD11076 Sep. 6, 2011 Oct. 5, 2011 Clear, however noticed separation in bulk 1,700 cps RD11077 Sep. 6, 2011 Oct. 5, 2011 Clear 1,600 cps RD11078 Sep. 6, 2011 Oct. 5, 2011 Clear and fluid 2,700 cps RD11079 Sep. 6, 2011 Oct. 5, 2011 Added 0.3% H2O to RD11076 3,500 cps Clear, fluid RD11080 Sep. 6, 2011 Oct. 5, 2011 Added 0.3% H2O to RD11077 3,900 cps Clear, fluid RD11081 Sep. 6, 2011 Oct. 5, 2011 Added 0.3% H2O to RD11078 2,600 cps Clear, fluid RD11085 Sep. 14, 2011 Oct. 5, 2011 Original test 4,800 cps 4,900 cps Thick and clear RD11086 Sep. 20, 2011 Oct. 5, 2011 Addition of 0.3% H2O to RD11085 = 5,200 cps 5,180 cps original Thick gel and clear

TBS1A 8% Formulation/Composition

TABLE 3 Active SiO2 % Trial micronized Castor oil Labrafil PVP grade DMI TransbutolP HPC Nisso C = Cabosil number % % % % % % % A = Aerosil200 RD11087 8 55.9 0000000 0000000 27  6  N − L = 0.2 A = 2.6 20 + 7 N − M = 0.3 RD11088 8 same 0000000 0000000 same same same Same plus (0.3% H2O) RD11089 8 46.5 0000000 K17 = 3 25 10 N − M = 0.5 C = 5   S630 = 2  RD11089A 8 same 0000000 same same same same Same plus (0.3% H2O) RD11090 8 39.0 0000000   K17 = 5.0 32 12  N − H = 0.3 C = 3.5 N − M = 0.2 RD11100 8 same 0000000 same same same same Added C = 2% for total of 5.5 RD11101 8 46.1 0000000   K17 =5.0 25 10  N − L = 0.4 C = 5.1 N − M = 0.4 RD11102 8 46.1 0000000   K17 = 5.0 25 10  N − L = 0.4 C = 5.1 plus N − M = 0.4 Addition of 1% for total of 6.1 RD11103 8 46.1 0000000   K17 =5.0 25 10  N − L = 0.4 C = 5.1 plus N − M = 0.4 addition of 0.3% water RD11104 8 42.2 4.0   K17 = 5.0 25 10  N − L = 0.4 A = 5.0 N − M = 0.4 RD11105 8 same same same same same same A = 5.0 addition of 0.5% total 5.5%

Process Outline for Active Trials: Lot # RD11087

Trial was initiated without PVP to identify impact on T solubility. The active dispersion in % DMI used did not provide a clear solution and did not clear up when adding to the Castor Oil/SiO2 mix. Even the co-solvents present in the HPC mixture did not provide a clear bulk Gel. To the HPV mixture 0.1% SiO2 was added to reduce stringing and stickiness.

Viscosity at 4,400

This trial however will be selected for the Franz Cell test to identify diffusion rate eliminating PVP.

Lot # RD11088

0.3% water was added to a portion of Lot RD11087 to identify impact on viscosity. As observed on 4% trials, increase in viscosity is not evident on the bulk mixed with SiO2 in the HPC. This trial not considered for F/C.

Lot # RD11089

This trial used the same quantitative formulation as the IMP Clinical 8%, however using an alternate source of HPC (original HPC source Klucel HF). Also made minor process changes, dissolved PVP in DMI only and added active. HPC was prepared in Transcutol and added to main bulk separately.

Obtained a clear solution when adding the active co-solvent mixture into the Castor-oil and no significant stringing with the addition of the HPC after addition of SiO2.

Viscosity of Gel on day of manufacture was 1,800 cps, when retested after 24 hours, 3,700 and after 48 hours up to 4,300. The re-test on October 3 (see table) recorded 4,500 cps.

This trial was selected for F/C test

Lot # RD11089A

0.3% water was added to a portion of Lot RD11089 to identify impact on viscosity.

Viscosity change over time similar to above trial, day of manufacture 2,700 cps, when retested after 24 hours, 3,920 and after 48 hours up to 4,600. The re-test on October 3 (see table) recorded 5,040 cps.

Selected for study on impact of water

Lot # RD11090

Used higher percentage of DMI and Transcutol to be split for various pre-mixes, similar with SiO2 to be added HPC. Made a pre-mix of Castor oil and SiO2, however due to the lower ratio between the 2 excipients, the mixture became quite thick and further thickened up when adding part of the DMI.

Did finish off the trial, ended up at low viscosity, day of manufacture 900 cps, test October 03-1,260 cps. Lower level of SiO2 was considered for study impact, however considering the processing issue (see RD11100)

not suitable for F/C test

Lot # RD11100

Using a portion of above trial RD11090, added an additional 2% SiO2 (for total of 5.5%) to study impact on Viscosity. Increased to 1,900 cps on day of manufacture and retest October 03 (see table) resulted in a value of 3.060

Lot # RD11101

To potentially reduce the impact of PVP, required to dissolve the active, during the addition to the Castor oil/SiO2 mixture, added 2% of SiO2 to the DMI-PVP-Testosterone mix, obtaining a viscous mix. After addition of that mixture to a dispersion of Castor oil containing 1% SiO2, maintained a viscous mixture at the temperature of 50% (would thicken up further on cooling). Further increase in viscosity with the addition of the HPC mix and final amount of SiO2.

Viscosity after cooling Gel to 21 C was 3,800 cps. (note that re-testing over time will be required, batch manufactured October 03)

This trial selected for F/C

Lot # RD11102

With the target for a 5,000 cps viscosity for the TBS1A project, the above RD11101 was so far the best candidate to evaluate impact of further addition of SiO2, hence to a portion of that lot additional 1% SiO2 was added. The rational for 6% was to obtain the same ratio of active to SiO2 as the targeted level of 3% SiO2 for the 4% strength.

Viscosity increase to 8,000 cps, this lot was selected for F/C study to identify impact of viscosity on rate of diffusion compared to RD11101 of same composition with exception of 1% addition in SiO2, may need to consider on assay obtained.

Lot # RD11103

Addition of water for impact on viscosity, not considered for follow up testing (see viscosity table for results, increase to RD11101 from 3,800 to 4,500 cps)

Lot # RD11104

Included this trial to evaluate addition of Labrafil. Labrafil was added to the Castor Oil mixed with SiO2 at 1%. As observed previously, addition of Labrafil to the Castor oil containing SiO2 increases viscosity. All other mixture prepared and added as per trial RD11101, with addition of 2% SiO2 to complete mixture. This mixture contains a larger percentage of air bubbles, common on formulations containing Labrafil. Viscosity obtained of 3,300 cps, will be followed up and tested at various time points.

Selected for F/C testing.

Lot # RD11105

Added to RD11104 an additional 0.5% SiO2 (% adjusted to avoid high increase observed on RD11102)

Increase from 3,300 to 4,100 cps

Not selected for F/C test

Note: Placebo trials are drawn up to identify impact on viscosity using the 2 different sources for Castor Oil and SiO2. These trials will also answer potential questions related to TBS1 and TBS2.

TABLE 4 TBS1A 8% strength Viscosity values using spindle #6, 20 rpm, Franz Cell = F/C Trial Manuf Lot number date Test date and values Comments RD11087 Sep. 20, 2011 Oct. 3, 2011 No PVP, solution not clear, 2.6% SiO2 4,400 cps Selected for Franz Cell RD11088 Sep. 20, 2011 Oct. 3, 2011 Added 0.3% H2Oto RD11087 4,040 cps RD11089 Sep. 25, 2011 Oct. 3, 2011 Based on original IMP, change in HPC 4,500 cps source and minor process step changes Selected for Franz Cell RD11089A Sep. 25, 2011 Oct. 3, 2011 As RD11089 plus 0.3% H2O 5,040 cps Selected for Franz Cell RD11090 Sep. 26, 2011 Oct. 3, 2011 3.5% SiO2 1,260 cps Potential for F/C RD11091 Sep. 26, 2011 Oct. 3, 2011 Added 0.3% H2O to RD11090 RD11100 Sep. 26, 2011 Oct. 3, 2011 Added to RD11090 to reach 5% SiO2 3,060 cps content RD11101 Oct. 3, 2011 Oct. 4, 2011 5% SiO2 3,800 cps Selected for Franz Cell RD11102 Oct. 4, 2011 Oct. 4, 2011 6% SiO2 8,000 cps Selected for Franz Cell RD11103 Oct. 4, 2011 Oct. 4, 2011 0.3% with 5% SiO2 4,500 cps RD11104 Oct. 4, 2011 Oct. 5, 2011 Includes 4% Labrafil, same comp for 3,300 cps polymers as RD11101 (air-bubbles) Selected or Franz Cell RD11105 Oct. 5, 2011 Oct. 5, 2011 Added additional 0.5% of SiO2 4,100 cps to RD11104

Pre-Mix RD Trials (Used for Addition in Active Trials)

TABLE 5 Trial #/ observation test Evaluation Composition Results/comments Used in RD trial # EV001A (pg 41) Dissolving HPC DMI-100 g Low viscosity grade Not transferred for Nisso grade M Transcutol P 50 g Stored for hydration use to RD trials Nisso HPC M-2.5 g 72 hrs Suitable viscosity for further additions EV001B (pg 41) Dissolving HPC DMI-100 g high viscosity grade Not transferred for Nisso grade H Transcutol P 50 g Stored for hydration use to RD trials Nisso HPC H-2.5 g 72 hrs Viscosity too high EV002A (pg 41) Dispersing Cabosil in DMI-125 g Obtained clear and Not transferred for DMI (purpose to Cabosil 10 g viscous dispersion use to RD trials study impact on Ratio related to viscosity in final Gel) Castor oil/Cabosil EV002B (pg 41) Dispersing Cabosil in Transcutol P 250 g Obtained no Not transferred for Transcutol P Cabosil 20 g increase viscosity, use to RD trials (purpose to study Ratio related to Solution milky in impact on viscosity Castor oil/Cabosil appearance in final Gel) RD11047 A Addition of PVP K17 DMI-100 g Suitable for Used in RD trial for in DMI only. PVP K17 15 g additional mixing addition of HPC-H Ratio represents 3% with HPC H and and active (see of PVP based on active. Note: used RD1150 and final Bulk Gel higher viscosity HPC RD1150A) formula grade with lower viscosity PVP grade RD11047B Addition of PVP DMI-100 g Suitable for Used in RD trial for K29/32 in DMI only. PVP K29/32 15 g additional mixing addition of HPC-M Ratio represents 3% with HPC M and and active (see of PVP based on active. Note: used RD1151 and final Bulk Gel lower viscosity HPC RD1151A) formula grade with higher viscosity PVP grade RD11047C Addition of PVP K90 DMI-100 g Not suitable to add Used in RD trial in DMI only. PVP K90 15 g any grade HPC, without HPC Ratio represents 3% however suitable to addition RD11056 of PVP based on add the active final Bulk Gel portion. formula RD11048 A Addition of PVP K17 DMI-80 g Suitable for Used in RD trial for in DMI and Transcutol P 20 g additional mixing addition of HPC-H Transcutol P PVP K17 15 g with HPC H and and active (see Ratio represents 3% active. Note: used RD11053 of PVP based on higher viscosity HPC final Bulk Gel grade with lower formula viscosity PVP grade RD11048B Addition of PVP DMI-80 g Suitable for Used in RD trial for K29/32 in DMI and Transcutol P 20 g additional mixing addition of HPC-M Transcutol P. PVP K29/32 15 g with HPC M and and active (see Ratio represents 3% active. Note: used RD11054 of PVP based on lower viscosity HPC final Bulk Gel grade with higher formula viscosity PVP grade RD11048C Addition of PVP K90 DMI-100 g Not suitable to add Used in RD trial in DMI and PVP K90 15 g any grade HPC, without HPC Transcutol P Ratio represents 3% however suitable to addition RD11055 of PVP based on add the active final Bulk Gel portion. formula RD11067 Prep of HPC in TP = 40 g Used in RD11076 Transcutol P only N—H = 0.75 g RD11068 Prep of HPC in TP = 40 g Used in RD11077 Transcutol P only N—H = 1.0 g RD11069 Prep of HPC in TP = 40 g Used in RD11078 Transcutol P only N—H = 0.5 g N—M = 0.5 g RD11075 Prep of base Castor oil/ solution used Aerosil200/ RD11076/RD11077/ DMI/ RD11078 PVP K30 Details in Table 2 Testosterone

Placebo TBS1A Trials

TABLE 6 Trial lot # Evaluation Composition Results/comments RD11032 Evaluate change in Labrafil M 1944 CS-500 g Viscosity 10,460 cps viscosity using Labrafil Cab-O-Sil - - - 40 g versus Castor Oil Cr 0 RD11033 Evaluate change Castor Oil - - - 500 g Viscosity 14 460 cps viscosity adding Cabosil Cab-O-Sil - - - 40 g first in Castor Oil Cr 0 Note: ratio used in IMP RD11034 Impact on adding DMI and RD11032-270 g Viscosity reduced to 8,740 Transcutol to mixture DMI-125 g RD11032 Transcutol P 50 g RD11035 Impact on adding DMI and Impact on adding DMI and Viscosity reduced to 3,600 Transcutol to mixture Transcutol to mixture RD11033 RD11032 RD11036A Mixture of Castor Oil and Castor oil . . . 125 g High viscosity out of range Labrafil, adding Cabosil Labrafil . . . 125 g followed by Cabosil . . . 20 g DMI/Transcutol P DMI . . . 125 g Transcutol P 50 g RD11036B Mixture of Castor Oil and Castor oil 0 . . . 125 g Viscosity 7,680 cps Labrafil followed by Labrafil . . . 125 g DMI/Transcutol P, add Cabosil . . . 20 g Cabosil last DMI . . . 125 g Transcutol P 50 g RD11043 Castor oil and Cab0sil, Castor oil 0 . . . 285 g followed by mixture of Cabosil . . . 20 g DMI/Transcutol P and DMI . . . 100 g HPC H Transcutol P 50 g HPC H . . . 2.5 g RD11043 Castor oil and Cab0sil, Castor oil 0 . . . 285 g followed by mixture of Cabosil . . . 20 g DMI/Transcutol P and DMI . . . 100 g HPC M and PVP K17 Transcutol P 50 g HPC M . . . 2.5 g PVP K15 . . . 15 g RD11057P TBS-2 Placebo for — — Analytical Lab Method RD11058P Castor oil an Cabosil A to D represents % RD11058P = 2740 cps A-B-C-D-E-F Mix followed by addition Labrafil of 2-4% with Part A 2% = 11,400 of Labrafil change in viscosity Part B 3% = 14,000 E impact of adding Oleic Part C 3.5% = 14,440 acid Part D 4% = 14,900 F impact of adding DMI to Part E with Oleic = 1,520 RD11058-A Part F-10% DMI to part A = 13,500 cps (incr. from 11,400) RD11083P Purpose of trial to HPC mix prep of Viscosity of base prior to decrease stringing and DMI/TranscutolP solvents addition of HPC mixture stickiness of HPC mixture plus Nisso HPC L and M was 5,300 cps, after when adding to base mix Solvated for 48 hours addition of HPC mixture of castor oil/Aerosil and followed by addition of (no stringing DMI SiO2 RD11084P Used part of RD1108P to add 0.3% H2O to evaluate impact on viscosity

Example 10 Franz Cell Studies—Testosterone Rates of Diffusion

Generally speaking, soak the membrane for 30 minutes in the diffusion solution. After put the membrane on the Franz Cell. Put the ring and the donor chamber on the membrane and clamp it. Add approx. one gram of gel (TBS 1 A 4% or 8%). Check the level of diffusion solution in Franz Cells. It's supposed to be on the mark. Put “parafilm” on the sampling port to avoid evaporation. Withdraw 0.3 mL of sample at 60, 120, 180, 240, 300 and 360 minutes using syringe. Add diffusion solution to make up to the mark of Franz Cells. Each sample should be collected in insert.

A typical Fanz cell used in accordance with this Example 9 and the invention is depicted in FIG. 12. The materials include:

Diffusion solution: Ethanol/Water 50:50

Membrane: Millipore 0.45 μm.

Temperature: 37°±0.5° C.

Stirring speed: 600 rpm.

Medium volume: 20 mL.

Surface area: 1.7671 cm²

Number of Franz Cells: 6.

Sampling time (minutes): 60, 120, 180, 240, 300 and 360.

Aliquot volume: 0.3 mL.

Insert: 0.4 mL.

The TBS1A formulations are as follows and as reported in the Examples above and herein. The rate of diffusion results of testosterone through the Franz cell membrane, normalized for each gel concentrations being tested, measured as slope/mgT %, are reported below in the Franz Cell Table.

4% TBS1A Trial formulations used in Franz Cell Raw Materials/grade % Process comments Trial Lot # RD11063 Batch size 500 g 24 hr Franz Cell Testosterone micronized 4.0 12% DMI to disperse PVP and active Castor Oil (V-O) 70.8 4% SiO2 in Castor oil plus 6% of DMI Steps: PVP K17 1.5 A: add all SiO2 to Castor Oil PVP K30 1.5 Followed by DMI portion PVP K90 0.0 B: to the DMI add PVP, follow Co PVP S630 0.0 With HPC and hold 24 hrs DMI 18.0 C: add active Transcutol P 0.0 D: add to mix A) HPC Nisso L 0.0 HPC Nisso M 0.0 Temp range NMT 60 C. HPC Nisso H 0.2 Homogenize active mixture SiO2 (Cabosil-Aerosil 200) 4.0 Viscosity 3,650 cps Oct. 5, 2011) Trial Lot # RD11085 Batch size 500 g 24 hrs Franz Cell Testosterone micronized 4.0 10% DMI used to dissolve active Castor Oil (V-O) 70.7 2.5% of SiO2 mixed into Castor Oil Steps: PVP K17 0.0 — A: Active /DMI mixture added PVP K30 0.0 — to Castor Oil/SiO2 mix PVP K90 0.0 — B: add SiO2 to HPC after 24 h Co PVP S630 0.0 — DMI 16.0 6% DMI used for HPC dispersion C: add HPC mixture to main Transcutol P 6.0 Used to disperse HPC and solvate for 24 hrs bulk HPC Nisso L 0.2 0.3% of SiO2 mixed into HPC mixture HPC Nisso M 0.3 Temp range NMT 60 C. HPC Nisso H 0.0 Homogenize active mixture SiO2 (Cabosil-Aerosil 200) 2.8 Viscosity 4,900 cps (Oct. 5, 2011) Trial Lot # RD11038 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Add to PVP mixture Castor Oil (V-O) 57.0 All Cabosil into Castor Oil A: add to the Castor Oil/SiO2 PVP K17 3.0 Mix the PVP active mixture PVP K30 0.0 PVP K90 0.0 Co PVP S630 2.0 DMI 20.0 All DMI and Transcutol P to disperse PVP Transcutol P 10.0 HPC Nisso L 0.0 HPC Nisso M 0.0 Homogenize active mixture HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 4.0 Viscosity 1,800 cps Trial Lot # RD11039 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Castor Oil (V-O) 29.0 Mix Castor oil + Labrafil + Cabosil PVP K17 3.0 PVP K30 0.0 PVP K90 0.0 Co PVP S630 2.0 PVP into DMI + Tr-P followed by active DMI 20.0 Transcutol P 10.0 Labrafil 29.0 HPC Nisso M 0.0 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 3.0 Viscosity 1,380 Trial Lot # RD11040 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Mix in 12% DMI and 6% Tr-P Castor Oil (V-O) 57.0 Combine Castor oil + SiO2 + 13% DMI + 4% TrP PVP K17 0.0 PVP K30 0.0 PVP K90 0.0 Co PVP S630 0.0 DMI 25.0 Transcutol P 10.0 HPC Nisso L 0.0 HPC Nisso M 0.0 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 4.0 Viscosity 11,040 Trial Lot # RD11042 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Active dissolve in 13% DMI + 4% Tr-P Castor Oil (V-O) 29.0 Castor oil + Labrafil + SiO2 + 12% DMI + 6% Tr-P PVP K17 0.0 PVP K30 0.0 PVP K90 0.0 Co PVP S630 0.0 DMI 25.0 Transcutol P 10.0 Labrafil 29.0 HPC Nisso M 0.0 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 3.0 Viscosity 1,430 cps Trial Lot #RD11051 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 20% DMI + PVP + N − M + 0.2% iO2 Castor Oil (V-O) 66.7 Castor Oil + SiO2 1.8% + 4% DMI PVP K17 0.0 Easier addition of HPC adding PVP K30 3.0 Small % of SiO2 PVP K90 0.0 Co PVP S630 0.0 DMI 24.0 Transcutol P 0.0 HPC Nisso L 0.0 HPC Nisso M 0.3 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 2.0 Viscosity 2,100 cps Trial Lot # RD11055 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 DMI 16% + Transc 4% + pvp + active Castor Oil (V-O) 62.0 Castor Oil + SiO2 3% + 7% DMI + Trans 1% PVP K17 0.0 PVP K30 0.0 PVP K90 3.0 Co PVP S630 0.0 DMI 23.0 Transcutol P 5.0 HPC Nisso L 0.0 HPC Nisso M 0.0 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 3.0 Exceeded test range Trial Lot # RD11078 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Castor Oil (V-O) 68.0 Castor oil + SiO2-3% + 6% DMI To be corrected to 67.8% PVP K17 0.0 for repeat (base) PVP K30 1.0 DMI 10% + pvp + active Base prep RD11075 PVP K90 0.0 Co PVP S630 0.0 DMI 16.0 Transcutol P 8.0 Transc P + both HPC Prep on RD11069 HPC Nisso L 0.0 HPC Nisso M 0.1 Requires adjustment of HPC Nisso H 0.1 Castor oil by 0.2% SiO2 (Cabosil-Aerosil 200) 3.0 Viscosity 2,700 cps Trial Lot #RD11054 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Castor Oil (V-O) 61.4 Castor Oil + SiO2 3% + DMI 7% + Transc 1% PVP K17 0.0 PVP K30 3.0 DMI 16% + Trans 4% + pvp + HPC + active PVP K90 0.0 Co PVP S630 0.0 DMI 23.0 Transcutol P 5.0 HPC Nisso L 0.0 HPC Nisso M 0.6 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 3.0 Viscosity 14,000 cps Trial Lot #RD11061 Batch size 500 g 6 hr Franz Cell Testosterone micronized 4.0 Castor Oil (V-O) 71.0 Castor oil + SiO2 + Labrafil PVP K17 2.0 DMI 16% + Transc 2% + PVP + active PVP K30 0.0 PVP K90 0.0 Co PVP S630 0.0 DMI 16.0 Transcutol P 2.0 Labrafil 2.0 HPC Nisso M 0.0 HPC Nisso H 0.0 SiO2 (Cabosil-Aerosil 200) 3.0 Viscosity 960 cps

TABLE 2 TBS1A 4% strength Viscosity values using spindle 6, 20 rpm, Repeat test ref to Franz Cell: F/C Trial Manuf Lot number date Test date and values Comments RD11037 Jul. 15, 2011 Oct. 4, 2011 Clear solution, previous results in July 620 cps   940 cps and follow up test Sep. 15, 2011 was 900 cps RD11038 Jul. 15, 2011 Oct. 4, 2011 Clear solution, original test 1,180 cps, follow up 1,800 cps Sep. 15, 2011 1,660 cps RD11039 Jul. 20, 2011 Oct. 4, 2011 Clear solution, previous results in July 980 cps 1,380 cps and follow up test Sep. 15, 2011 was 1,300 cps RD11040 Jul. 20, 2011 Oct. 4, 2011 Clear Gel, previous results in July 10,400 cps 11,040 cps  and follow up test Sep. 15, 2011 was 10,140 cps RD11041 Jul. 21, 2011 Oct. 4, 2011 Clear solution, previous results in July 500 cps 1,420 cps and follow up test Sep. 15, 2011 was 1,500 cps RD11042 Jul. 21, 2011 Oct. 4, 2011 Clear solution, test Sep. 15, 2011 was 1,720 cps 1,430 cps RD11050 Aug. 9, 2011 Oct. 4, 2011 Original comment sticky mixture, Sep. 15, 2011 Test not valid results 2,460 Do not use trial lot for F/C Poor mixture, HPC settled to bottom as a slug RD11050A Aug. 9, 2011 Oct. 4, 2011 Original comment sticky mixture, results Test not valid Sep. 15, 2011 3,000 cps (increased during test from 2,400) Do not use trial lot for F/C Poor mixture, HPC settled to bottom as a slug RD11051 Aug. 9, 2011 Oct. 4, 2011 clear, results Sep. 15, 2011 1,940 cps 2,100 cps▴ Note: viscosity values increase during 30 sec test RD11051A Aug. 9, 2011 Oct. 4, 2011 clear, results Sep. 15, 2011 2,560 cps 2,540 cps▴ Note: viscosity values increase during 30 sec test RD11053 Aug. 10, 2011 Oct. 4, 2011 Clear but sticky with air bubbles, results 4,500 cps▴ Sep. 15, 2011 4,060 cps Note: viscosity values increase during 30 sec test RD11054 Aug. 10, 2011 Oct. 4, 2011 Sep. 15, 2011 test HPC globules, 15,000 cps 14,000 cps▴  Do not use trial lot for F/C, Note: viscosity values increase during 30 sec test Build up of HPC on spindle RD11055 Aug. 10, 2011 Sep. 15, 2011, EEEEEE

The TBS-1A Gel In Vitro Release Rate Validation concerning Release Rate Study Summary for TBS-1A Gel 4.0% and TBS-1A Gel 4.5% are presented in Exhibits A and B submitted herewith.

These summaries summarize the release rate experiment data for exemplary TBS-1A Gels. There are four Nasobol Gels (0.15%, 0.6%, 4.0% and 4.5%) for the method validation. The purpose of the Day1 and Day2 test are to determine the specificity and intraday/interday precision of the slope(release rate), Day3 and Day4 are to evaluate the slope sensitivity to the sample strength variation.

See Exhibit A (4.0%) and Exhibit B (4.5%) submitted herewith, both of which are incorporated herein by reference in their entireties.

Example 11 In Vitro Release Rate (Ivrt) Comparison Testing

IVRT experimental approach is used for comparison of products in semi-solid dosage form through evaluation of the drug release. In order to have fair comparison, products to be compared should be of comparable age and their release rates should be determined on the same day, under the same conditions. To ensure an unbiased comparison, sample position within the bank of Franz cells are randomized. The test (T) product and reference (R) product in each run is randomized or pre-assigned in a mixed arrangement.

Method Parameter Main Alternate parameters Franz Cells Franz Cells membrane: durapore 0.45 μm, membrane: durapore 0.45 μm, HVLP02500 HVLP02500 ring diameter 15 mm diamèter 15 mm surface: 1.767 mm″ surface: 1.767 mm″ thickness: 3.2 mm thickness: 1.63 mm Gel Volume: 565.44 mm″ gel Volume: 288.02 mm″ receiving media volume: 12 ml Volume media recptor: 7.5 ml Ethanol Water 50/50 ETOH/water 50/50 600 rpm 600 rpm Assay Assay UPLC HPLC Concentrations from 3 Concentrations 5 μg/ml to 100 μg/ml μg/ml to 200 μg/ml

The slope comparison test recommended by the FDA is performed and provides the evidence of the reproducibility of the IVRT method.

The two different formulations of the testosterone gel products, Table 1, are applied on 12 cells of the modified Franz-Cell apparatus system: 6 cells for reference product (R) and 6 cells for test product (T), as depicted in FIG. 13. The two gel products, Testosterone Nasabol Gel 4%, lot # E10-007, and TBS1A Testosterone Nasal Gel 4%, lot # IMP 11002, are described in Example 6 and designated as 4% TSA-1A and TBS1.

TABLE 1 TBS-1A 4% Material TBS1 (A) Dimethyl isosorbide 0 25.0 Diethyleneglycol ethyl 0 10.0 ether Povidone 0 3.0 Copovidone 0 2.0 Hydroxypropyl 0 0.5 cellulose Testosterone 4.0 4.0 micronized Castor oil 88.0 50.5 Labrafil M1944CS 4.0 0 Colloidal silicon 4.0 5.0 dioxide Water 0 0 Total 100.0 100.0

Samples are collected at 1, 2, 3, 4, 5 and 6 hours and are tested.

Franz Cell Apparatus Position Layouts for Comparison Testing

The Release Rates (slope) from the six cells of T-product and from the other six cells of the R-product are obtained. A 90% Confidence Interval (CI) for the ratio (T/R) of median release rates is computed.

A table with six rows and seven columns is generated and reference slopes (RS) are listed across the first row and test slopes (TS) are listed down the first column of Table 2. Individual T/R ratios (30) between each test slope and each reference slope are computed and the corresponding values are entered in the table.

TABLE 2 Calculation of T/R Ratios Slope RS1 RS2 RS3 RS4 RS5 RS6 TS1 TS 1/RS 1 TS 1/RS2 TS 1/RS3 TS 1/RS4 TS 1/RS5 TS 1/ RS6 TS2 TS2/RS 1 TS2/RS2 TS2/RS3 TS2/RS4 TS2/RS5 TS2/ RS6 TS3 TS3/RS 1 TS3/RS2 TS3/RS3 TS3/RS4 TS3/RS5 TS3/ RS6 TS4 TS4/RS I TS4/RS2 TS4/RS3 TS4/RS4 TS4/RS5 TS4/ RS6 TS5 TS5/RS 1 TS5/RS2 TS5/RS3 TS5/RS4 TS5/RS5 TS5/ RS6 TS6 TS6/RS 1 TS6/RS2 TS6/RS3 TS6/RS4 TS6/RS5 TS6/ RS6

These 30 T/R ratios are ranked from lowest to highest. The sixth and twenty-fifth ordered ratios represent low and upper limits of the 90% CI for the ratios of median release rates.

Standard Criteria:

Test and reference product are considered to be the same if the 90% CI falls within the limits of 75%-133.3%.

Two batches of Testosterone Nasabol Gel 4%, lot # E10-007, and TBS1A Testosterone Nasal Gel 4%, lot # IMP 11002, are tested and evaluated for sameness.

A statistical comparison is carried out by taking the ratio of release rates from 6 cells of the reference lot # E10-007 (R) against 5 cells of the test batch lot # IMP 11002 (T).

During the in vitro drug releases test, the reference batch and the test batch are applied in a randomized manner on the cells on Apparatus A and B of the modified Franz Cell System.

Release Rate (slope) from five cells of the test product (T) and six cells of the reference product (R) are compared. A 90% Confidence Interval (CI) for the ratio (T/R) of median release rates is computed.

The 90% Confidence Interval is represented by the sixth and twenty-fifth Release Rate ratios when ranked from lowest to highest. These ratios correspond to 160.77% and 202.90% respectively and do not meet the limits for sameness (CI 75%-133.33%). Therefore, the two batches of Testosterone Nasabol Gel 4%, lot # E10-007 and TBS1A Testosterone Nasal Gel 4%, lot # IMP 11002 are not considered the same.

Two gel products, Testosterone Nasabol Gel 4%, lot # E10-007, and TBS1A Testosterone Nasal Gel 4%, lot # IMP 11002, are tested and evaluated for sameness. The Mean Release Rate (slope) for the Test lot # IMP 11002 is about 1.8 times higher than for the Reference lot # E10-007. The two tested products are found to be not the same.

The In Vitro Release Rate (IVRT) testing results and raw data are in Tables 3-8 below and FIG. 23.

TABLE 4 4% Gel Release Rate Comparison Testosterone TBS1A Testosterone Nasal Gel 4% Test Lot# IMP11002 Concentration of Active (μg/mL) versus Time Amount Released (μg/mL) Calculation by Linear Regression Curve Time Cell A#1 Cell A#3 Cell A#5 Cell B#2 Cell B#4 Cell B#6 Mean 1-5 % RSD 1-5  60.00 118.922 115.401 122.547 123.279 118.672 114.557 118.896 3.0 120.00 195.377 182.991 201.133 205.222 191.880 — 195.321 4.4 180.00 280.637 246.686 274.856 282.957 264.605 261.063 268.434 5.1 240.00 344.420 291.933 329.143 346.540 317.162 324.788 325.664 6.2 300.00 401.961 330.531 376.137 403.828 369.302 388.545 378.717 7.2 360.00 462.471 379.994 426.269 462.433 417.526 445.583 432.713 7.3 Actual Amount of Active Released (μg/cm²) versus Time^(0.5) Amount Released (μg/cm²) Cell A#1 Cell A#3 Cell A#5 Cell B#2 Cell B#4 Cell B#6 Mean 1-5 % RSD 1-5 Time^(0.5)  7.75 807.574 783.664 832.191 837.181 805.876 777.932 807.400 3.0 10.95 1360.413 1275.306 1400.526 1428.501 1336.594 #N/A 1360.266 4.4 13.42 1994.677 1759.622 1956.716 2014.450 1884.747 #N/A 1922.042 5.4 15.49 2507.220 2136.684 2404.439 2526.291 2316.520 #N/A 2376.231 6.7 17.32 2995.422 2451.397 2630.278 3013.375 2670.332 #N/A 2816.161 7.7 18.97 3520.067 2910.814 3277.707 3525.812 3192.305 #N/A 3285.301 7.8 Slope 242.85 187.78 217.83 239.55 213.29 220.26 10.1  R² 0.9912 0.9947 0.9958 0.9927 0.9942 0.9937 0.2

Tables 4 and 5 are graphically represented in FIGS. 38 and 39 respectively.

TABLE 5 4% Gel Release Rate Comparison Testosterone Nasobol Gel 4% Gel Reference Lot# E10-007 Concentration of Active (μg/mL) versus Time Amount Released (μg/mL) Calculation by Linear Regression Curve Time Cell A #2 Cell A#4 Cell A#5 Cell B#1 Cell B#3 Cell B#5 Mean 1-6 % RSD 1-6  60.00  96.792 104.726 101.499  98.956  96.994 101.074 100.341 2.7 120.00 143.746 153.402 151.866 148.611 146.111 152.389 149.356 2.6 180.00 181.187 191.204 190.149 185.651 182.536 188.818 186.591 2.2 240.00 206.803 219.307 216.557 214.046 212.670 218.650 214.672 2.2 300.00 234.373 243.717 243.534 239.656 238.437 241.174 240.165 1.5 360.00 253.244 262.615 261.716 259.500 255.210 263.639 259.321 1.6 Actual Amont of Active Released (μg/cm²) versus Time^(0.5) Amount Released (μg/cm²) Cell A#2 Cell A#4 Cell A#6 Cell B#1 Cell B#3 Cell B#5 Mean 1-6 % RSD 1-6 Time^(0.5)  7.71 657.294 711.172 689.258 672.003 672.247 686.372 681.391 2.7 10.95 1003.536 1071.352 1060.212 1037.186 1020.219 1063.304 1042.635 2.6 13.42 1298.462 1371.463 1382.858 1330.766 1308.915 1353.934 1337.750 2.2 15.49 1523.682 1616.405 1596.093 1576.120 1565.197 1609.943 1581.240 2.2 17.32 1769.419 1844.221 1841.242 1810.596 1800.351 1824.766 1815.099 1.5 18.97 1963.883 2041.513 2032.959 2013.163 1981.718 2045.561 2013.135 1.7 Slope 116.80 119.04 120.10 119.69 118.02 129.59 119.04 1.2 R² 0.9998 0.9997 0.9996 0.9996 0.9992 0.9997 0.9995 0.0

TABLE 6 Comparison Study Franz Cell Release Rate Comparison R - Reference Lot# E10-007  Testosterone Nasabol Gel 4% Gel T - Test Lot# IMP 11002    TBS1A Testosterone Nasal Gel 4% R T 116.80 119.04 120.10 119.69 118.02 120.59 242.85 2.0792 2.0401 2.0221 2.0290 2.0577 2.0138 187.78 1.6077 1.5775 1.5635 1.5689 1.5911 1.5572 217.83 1.8650 1.8299 1.8137 1.8200 1.8457 1.8064 239.55 2.0509 2.0123 1.9946 2.0014 2.0297 1.9865 213.29 1.8261 1.7918 1.7759 1.7820 1.8072 1.7687 Note: Test Lot Vial# B#6 at 2 hour was missing injection. Comparison calculated by 5 × 6 = 30 individual T/R ratios, and the limits of 90% would be sixth and twenty-fifth order individual T/R ratios.

TABLE 7 Sixth Ordered Ratio: 160.77% Twenty-fifth Ordered Ratio: 202.90% Test and reference products are considered to be the “same” if the 90% CI falls within the limits of 75%-133.33%.

TABLE 8 Amount of Active Released (μg/cm²) Time^(0.5) Lot# IMP11002 Lot# E10-007 7.75 807.400 681.391 10.95 1360.268 1042.635 13.42 1922.042 1337.75 15.49 2378.231 1581.24 17.32 2816.161 1815.099 18.97 3285.301 2013.135

TABLE 9 In Vitro release Rate Testing Products: TBS1A Testosterone Nasal Gel 4% and Testosterone Nasobol Gel 4% Objective: Release rate comparison between the two testosterone gel products. Side Sample Information Release Rate Results Reference Testosterone Nasobol Gel 4% Average slope: 119.87 μg/cm² · min^(−0.5) Batch The reference Lot #E10-007 RSD of Slopes: 1.8% Expiry date: N/A R² of Lowest Linearity: 0.9995 Diteba Sample ID: CSB-SPL-00200 Number of Cells: 6 Position of Cells: System (1) A#2, #4, #6; System (2) B#1, #3, #5 Test TBS1A Testosterone Nasal Gel Average slope: 300.02 μg/cm² · min^(−0.5) Batch 4% RSD of Slopes: 9.3% The test batch (Lot #IMP R² of Lowest Linearity: 0.9995 11001) Expiry date: N/A Diteba Sample ID: CSB-SPL-00209 Number of Cells: 6 Position of Cells System (1) A#1, #3, #5; System (2) B#2, #4, #6 Comparison Results Release Rate Comparison Comparison Limits: 75.00% to 1.33.33% Stage One  8^(th) ordered ratio: 228.50% 29^(th) ordered ratio: 264.03% Stage Two 110^(th) ordered ratio: N/A 215^(th) ordered ratio: N.A

Example 12 A Phase-1 Open Label, Balanced, Randomized, Crossover, Two Groups, Two-Treatments, Two-Period, Pilot Study in Healthy Male Subjects

A phase-1 open label, balanced, randomized, crossover, two groups, two-treatments, two-period, pilot study in healthy male subjects to determine the feasibility of a multiple dose dispenser for testosterone intranasal gel as measured by pharmacokinetics

Testosterone replacement therapy aims to correct testosterone deficiency in hypogonadal men. Trimel BioPharma has developed an intranasal testosterone gel (TBS-1) as alternative to the currently available testosterone administration forms. To date, a syringe was used to deliver TBS-1 in clinical studies. Trimel identified a multiple dose dispenser intended for commercial use. The purpose of this study was to demonstrate the relative performance of the multiple dose dispenser in comparison to the syringe used previously in clinical trials.

This was an open label, balanced, randomized, crossover, two-group, two-treatment, two-period, pharmacokinetic study of TBS-1 testosterone nasal gel in healthy, male subjects aged 18 to 28. Treatment consisted of 4.5% TBS-1 testosterone gel as a single dose of 5.5 mg of testosterone per nostril, delivered using either a syringe or the multiple dose dispenser, for a total dose of 11.0 mg given at 21:00 hours. Prior to first administration, subjects were admitted to the unit for blood sampling in order to determine a baseline testosterone profile. Wash-out between drug administrations was at least 48 hours.

All subjects completed the study successfully and treatment was well tolerated.

The total exposure to testosterone as estimated by the mean area under the serum concentration-time curve (AUC₀₋₁₂ in ng·hr/dL), is higher after TBS-1 administration using the dispenser or syringe than endogenous levels alone (7484 and 7266, respectively, versus 4911 ng*h/dL. Mean C_(max) is higher after administration with the dispenser than after administration using a syringe (1028 versus 778.8 ng/dL, respectively). T_(max) occurs earlier following administration using the dispenser compared to the syringe (2.75 versus 5.6 hours, respectively. Thus, testosterone absorption seems to be faster with the multiple dose dispenser than with a syringe, but the total absorbed amount is similar. Also, in previous studies the syringe Tmax obtained in patient was closer to 1.0 or 2.0 hours.

When plotting probability density of the log ratio of testosterone levels reached with the multiple dose dispenser over levels reached with the syringe as shown in FIG. 3, no significant difference was demonstrated for either AUC₀₋₁₂ or C_(max) within the lower and upper limit of the 95% confidence intervals. There is a trend toward a difference for C_(max). However, this data does not confirm bioequivalence at a confidence interval level of 90% for either AUC₀₋₁₂ or C_(max). If the trends found here are confirmed in a larger data set, the routes of administration would be almost equivalent for AUC₀₋₁₂, but t for C_(max) further investigation may be required as the Cmax/tmax profile obtained in volunteers does not seem to match the one obtained in patients.

Testosterone as a Treatment for Hypogonadism

Endogenous androgens are responsible for the normal growth and development of the male sex organs as well as promoting secondary sex characteristics including the growth and maturation of the prostate, seminal vesicles, penis, and scrotum; the development of male hair distribution, such as beard, pubic, chest, and axillary hair, laryngeal enlargements, vocal cord thickening, alterations in body musculature, and fat distribution.

Hypogonadism in men is characterized by a reduced concentration of serum testosterone resulting in signs and symptoms that may include decreased libido, erectile dysfunction, decreased volume of ejaculate, loss of body and facial hair, decreased bone density, decreased lean body mass, increased body fat, fatigue, weakness and anaemia.

The causes of hypogonadism can be primary or secondary in nature. In primary hypogonadism (congenital or acquired) testicular failure can be caused by cryptorchidism, bilateral torsion, orchitis, vanishing testis syndrome, orchidectomy, Klinefelter's syndrome, chemotherapy, or toxic damage from alcohol or heavy metals. These men usually have low serum testosterone levels and serum gonadotropin levels (FSH, LH) above the normal range.

In secondary hypogonadism (Hypogonadotropic Hypogonadism (congenital or acquired)) the defects reside outside the testes, and are usually at the level of the hypothalamus or the pituitary gland. Secondary hypogonadism can be caused by Idiopathic Gonadotropin or LHRH deficiency, or pituitary hypothalamic injury from tumors, trauma, or radiation. These men have low serum testosterone levels but have serum gonadotropin levels in the normal or low ranges.

Testosterone hormone therapy is indicated as a hormone replacement therapy in males for conditions associated with a deficiency or absence of endogenous testosterone. The currently available options for administration of testosterone are oral, buccal, injectable, and transdermal.

Trimel BioPharma has developed an intranasal testosterone gel (TBS-1) as a hormone replacement therapy for the treatment of male hypogonadism. The nasal mucosa offers an alternative route of administration that is not subjected to first pass metabolism, has high permeability, with rapid absorption into the systemic circulation. The advantages of the testosterone intranasal gel when compared to other formulations include ease of administration and no transference of testosterone to other family members.

Investigational Medicinal Product

The investigational medicinal product in this trial was TBS-1, an intranasal testosterone dosage form. A description of its physical, chemical and pharmaceutical properties can be found in the Investigator's Brochure.

Summary of Non-Clinical and Clinical Studies Summary of Non-Clinical Studies

An overview of the pharmacology, toxicology and preclinical pharmacokinetics of different testosterone preparations and administration routes is provided in the Investigator's Brochure Product-specific repeat dose toxicity and tolerance studies have been performed in ex vivo models and in different animal species.

Summary of Previous TBS-1 Clinical Studies

To date, Trimel has completed four Phase II clinical trials in hypogonadal men. The most recently conducted study, TBS-1-2010-01, is described below and the other studies are summarized in the Investigator's Brochure.

The objective of study TBS-1-2010-01 is to examine the efficacy and tolerability of 4.0% and 4.5% TBS-1 testosterone gel in hypogonadal men. In this study, TBS-1 is administered using a syringe, not the commercial multiple dose dispenser. The doses and dosing regimens that were used in study TBS-1-2010-01 are described in Table 1 below.

The results from all treatment groups met the FDA criteria for efficacy; defined as that at least 75% of subjects should achieve an average total T concentration (C_(avg)) in the normal range, a 24 hour C_(avg) value ≥300 ng/dL and ≤1050 ng/dL.

TABLE 1 Summary of previous TBS-1 studies C_(avg) (% of subjects with C_(avg) within the Dosing regimen Total daily dose reference range)  13.5 mg of TBS-1 (4.5%) BID   27 mg/day  419 ng/dL (100%)  10.0 mg of TBS-1 (4.0%) TID   30 mg/day 413 ng/dL (87%) 11.25 mg of TBS-1 (4.5%) TID 33.75 mg/day 396 ng/dL (85%)

Summary of Benefits and Risks to Subjects Benefits

Testosterone replacement therapy for hypogonadal men should correct the clinical abnormalities of testosterone deficiency. Since this was a Phase I study enrolling normal healthy men between the ages of 18-45, for a short period of time, it was not anticipated that these volunteers would directly benefit by taking part in this study. Volunteers were financially compensated for their participation.

Risks

The risk to the subject by participating in this study was considered to be minimal. Testosterone replacement therapy is indicated for the treatment of hypogonadism and TBS-1 has been administered to over 100 men with minimal side effects.

As TBS-1 is an investigational drug that is in clinical development, the complete side effect profile was not fully known. Epistaxis, nasal congestion, nasal discomfort, nasal dryness and nasal inflammation have been reported following use of TBS-1. Side effects from approved (prolonged) testosterone replacement therapy include elevated liver enzymes (alanine aminotransferase, aspartate aminotransferase), increased blood creatine phosphokinase, increase in prostatic specific antigen, decreased diastolic blood pressure, increased blood pressure, gynecomastia, headache, increased hematocrit/hemoglobin levels, hot flushes, insomnia, increased lacrimation, mood swings, smell disorder, spontaneous penile erection, and taste disorder.

The main benefit of the intranasal drug delivery route is that with this method many of the different disadvantages observed with other products would not be expected. This would include skin-to-skin transfer, stickiness, unpleasant smell (gels), skin irritation (patches), elevated DHT (patches and oral), injection pain and high T and DHT peaks (intramuscular injection), food interaction (oral).

Trial Rationale

Trimel identified a multiple dose dispenser that was intended as the commercial dispenser to be used in this clinical trial program. To date, a syringe has been used to deliver TBS-1 in the previous clinical trials. The purpose of this study was to demonstrate the comparability of the pharmacokinetic results obtained with a multiple dose dispenser or a syringe.

REFERENCES

-   1. Nasobol® Investigator Brochure Release Date 19 Aug. 2010, Edition     No: 5. -   2.     http://www.androgel.com/pdf/500122-00127_Rev_1E_Sep_2009_FPI_with_MedGuide.pdf     (Last accessed on 6 Sep. 2010). -   3. http://www.mattern-pharmaceuticals.com/downloads/Nasobol.pdf     (Last accessed on 6 Sep. 2010). -   4.     http://www.medicines.org.uk/EMC/medicine/22159/SPC/Testim+Gel/(Last     accessed on 6 Sep. 2010).

Study Objectives

The primary study objective is to compare a pharmacokinetic profile of testosterone after administration of TBS-1 using two different dispensers in healthy male subjects.

The secondary objective is to assess the safety of TBS-1.

Investigational Plan Overall Study Design and Plan

This is an open label, balanced, randomized, crossover, two-group, two-treatment, two-period, pharmacokinetic study of testosterone nasal gel formulation in healthy, adult, male human subjects. The study event schedule is summarized in Section ?????in Table 2.

Healthy male volunteers, aged 18 to 45 years (inclusive) were screened for this study. The goal was to randomize 12 male subjects for the study.

There was a washout period of 6 days between each drug administration.

Discussion of Study Design

As this is a relatively small Phase I PK study with the intent to compare a pharmacokinetic profile of testosterone after administration of TBS-1 from two different dispensers in healthy male subjects, a true sample size calculation is not performed. Based on typical early-stage, pharmacokinetic studies, groups of 6 subjects per cohort are sufficient for an acceptable description of the pharmacokinetic parameters after single dose administration.

Selection of Study Population Inclusion Criteria

The following eligibility assessments have to be met for subjects to be enrolled into the study:

-   -   1. Healthy male human subjects within the age range of 18 to 45         years inclusive     -   2. Willingness to provide written informed consent to         participate in the study     -   3. Body-mass index of ≤35 kg/m²     -   4. Absence of significant disease or clinically significant         abnormal laboratory values on laboratory evaluations, medical         history or physical examination during screening     -   5. Normal otorhinolaryngological examination     -   6. Non-smokers for at least six months     -   7. Comprehension of the nature and purpose of the study and         compliance with the requirement of the protocol

Exclusion Criteria

A subject is not eligible for inclusion in this study if any of the following criteria applied:

-   -   1. Personal/family history of allergy or hypersensitivity to         testosterone or related drugs     -   2. Past history of anaphylaxis or angioedema     -   3. Any major illness in the past three months or any clinically         significant ongoing chronic medical illness e.g. congestive         heart failure, hepatitis, pancreatitis etc.     -   4. Presence of any clinically significant abnormal values during         screening e.g. significant abnormality of Liver Function Test         (LFT), Renal (kidney) Function Test (RFT), etc.     -   5. Hemoglobin <13 g/dl and Hematocrit >52% during screening     -   6. Any cardiac, renal or liver impairment, any other organ or         system impairment     -   7. History of seizure or clinically significant psychiatric         disorders     -   8. Presence of disease markers for HIV 1 and/or 2, Hepatitis B         and/or C virus     -   9. History of nasal surgery, specifically turbinoplasty,         septoplasty, rhinoplasty, (“nose job”), or sinus surgery     -   10. Subject with prior nasal fractures     -   11. Subject with active allergies, such as rhinitis, rhinorrhea,         or nasal congestion     -   12. Subject with mucosal inflammatory disorders, specifically         pemphigus, or Sjogren's syndrome     -   13. Subject with sinus disease, specifically acute sinusitis,         chronic sinusitis, or allergic fungal sinusitis     -   14. History of nasal disorders (e.g. polyposis, recurrent         epistaxis (>1 nose bleed per month), abuse of nasal         decongestants) or sleep apnea     -   15. Subject using any form of intranasal medication delivery,         specifically nasal corticosteroids and oxymetazoline containing         nasal sprays (e.g. Dristan 12-Hour Nasal Spray)     -   16. History of asthma and/or on-going asthma treatment     -   17. Regular drinkers of more than three (3) units of alcohol         daily (1 unit=300 ml beer, 1 glass wine, 1 measure spirit), or         consumption of alcohol within 48 hours prior to dosing and         during the study.     -   18. Volunteer demonstrating a positive test for alcohol         consumption (using breath alcohol analyzer) at the time of         check-in during the admission periods.     -   19. History of, or current evidence of, abuse of alcohol or any         drug substance, licit or illicit     -   20. Volunteers demonstrating a positive test for drugs of abuse         in urine (Opiates, Benzodiazepines, Amphetamines, THC and         cocaine) at the time of check-in during admission periods     -   21. Inaccessibility of veins in left and right arm     -   22. Receipt of any prescription drug therapy within four weeks         of the first admission period.     -   23. Difficulty in abstaining from OTC medication (except         occasional paracetamol/aspirin) for the duration of the study     -   24. Volunteers demonstrating serum PSA 4 ng/ml     -   25. Participation in any other research study during the conduct         of this study or 30 days prior to the initiation of this study.     -   26. Blood donation (usually 550 ml) at any time during this         study, or within the 12 week period before the start of this         study.         Removal of Patients from Therapy or Assessment

All 12 subjects who enroll, complete the study successfully, and no subjects are replaced.

Treatments Treatments Administered

For the drug administration, subjects are instructed on how TBS-1 is applied intranasally with the pre-filled syringes or the multiple dose dispensers. Self-administration of TBS-1 is monitored by the study personnel. Each subject is instructed not to sniff or blow his nose for the first hour after administration.

TABLE 2 Treatment schedule BASELINE PERIOD I PERIOD II Day 1/2 Day 2/3 Day 4/5 Subject Time Time Time GROUP number 21:00-09:00 21:00-09:00 21:00-09:00 A 1-6 12 hour baseline TREATMENT 1 TREATMENT 2 T profile B  7-12 12 hour baseline TREATMENT 2 TREATMENT 1 T profile

Treatment 1 consists of TBS-1 syringes that are pre-filled with 4.5% testosterone gel to deliver a single dose of 5.5 mg of testosterone per nostril, for a total dose of 11.0 mg that is administered at 21:00 hours (±30 minutes) on Day 2 of Period I for Group A and Day 4 of Period II for Group B.

Treatment 2 consists of a TBS-1 multiple dose dispensers that are pre-filled with 4.5% testosterone gel to deliver a single dose of 5.5 mg of testosterone per nostril, for a total dose of 11.0 mg that is administered at 21:00 hours (±30 minutes) on Day 2 of Period I for Group B and Day 4 of Period II for Group A.

Identity of Investigational Product(s)

The investigational product in this trial is TBS-1, an intranasal testosterone dosage form.

Study medication consists of TBS-1 gel and is packed either in a single use syringe that is designed to expel 125 μl of gel, with two syringes packaged per foil pouch, or in a multiple dose dispenser that is designed to expel 125 μl of gel/actuation.

Study medication is dispensed by the study pharmacist who prepares the individual study kits which contained two syringes in a pouch or the multiple dose dispenser.

Method of Assigning Patients to Treatment Groups

Treatment assignment is determined according to the randomization schedule at the end of Visit 1. Subjects who met the entry criteria are assigned randomly on a 1:1 basis to one of the two treatment groups (Group A or Group B). The randomization is balanced and the code is kept under controlled access. The personnel that are involved in dispensing of study drug is accountable for ensuring compliance to the randomization schedule.

Selection and Timing of Dose

As healthy males have endogenous testosterone levels that fluctuate with a circadian rhythm which peaks in the early morning, it is decided to dose the study medication at night.

Blinding

This is an open-label study for both the subjects and the investigator, as the physical differences in the intranasal dosing dispensers prevent blinding.

Prior and Concomitant Therapy

None of the subjects use prescription medication immediately prior to, during or the 2 weeks after the study. One subject receives a single dose of paracetamol (2 tablets of 500 mg) just before discharge on the morning after the baseline visit (before administration of any study medication). There are no other reports of medication use.

Treatment Compliance

All subjects receive both doses of study medication according to the instructions and are monitored by study personnel for one-hour post-dosing to assure conformity to the TBS-1 instructions. All subjects remain in the clinic during the 12-hour PK sampling time period; during which they are monitored closely.

Screening

The screening visit (visit 1) takes place at a maximum of 21 days before the first study day. After giving informed consent, the suitability of the subject for study participation is assessed at screening which consists of the following items:

-   -   Medical history     -   Physical examination and Vital Signs.     -   A fasting blood sample is taken to determine the following:         Complete Blood Count, Chemistry profile; testing for HBV, HCV,         HIV and PSA.     -   Urinalysis, urine drug screen, and Breath Alcohol Testing.     -   An otorhinolaryngological nasal endoscopic examination is         performed by an ENT specialist.

Subjects meeting all of the inclusion and no exclusion criteria are enrolled into the study and are randomized into one of two treatment groups (1 or 2).

Study Days

Subjects are admitted to the clinical research centre at 19:30 hours on Day 1 (Visit 2, baseline), 2 (Visit 3, Period 1) and 4 (Visit 4, Period 2). After check-in tests for drug-abuse and alcohol consumption are performed. Vital signs are recorded and subjects are questioned about changes in their health.

During Visit 2, a 12 hour baseline testosterone profile is measured. Blood for the 12 hour baseline testosterone profile is drawn according to the following schedule: first sample at 20:45 hours and then at 0.33, 0.66, 1.00, 1.50, 2.00, 3.00, 4.00, 5.00, 6.00, 8.00, 10.00, and 12.00 hours relative to 21:00 time point (a total of 13 samples). On Day 2 vital signs are measured and safety parameters (symptoms, AEs) recorded before check-out.

Dosing is performed on the evenings of Day 2 and 4, at 21:00 hr. Before dosing an ENT examination is performed and a pre-dose, baseline serum testosterone blood sample is drawn. After dosing, a 12 hour testosterone PK profile is measured. The blood samples are drawn according to the following schedule after the 21:00 hour dosing: 0.33, 0.66, 1.00, 1.50, 2.00, 3.00, 4.00, 5.00, 6.00, 8.00, 10.00, and 12.00 hr time points (a total of 13 samples per period).

On Day 3 and 5 vital signs are measured, ENT examination are performed and safety parameters are recorded (symptoms, AEs) after the last PK sampling and before check-out. On Day 5 a final examination is performed, consisting of a general physical examination and clinical laboratory investigation (Complete Blood Count, Chemistry profile and Urinalysis).

Pharmacokinetic Sampling

Blood samples for analysis of testosterone levels are collected in 4 ml standard clotting tubes using an intravenous cannula. Tubes are left to clot for 30-45 minutes. Samples are centrifuged within one hour at 2000 g for 10 minutes at 4° C. The serum is then transferred directly to two aliquots of 1 ml each and frozen at −40° C.

Safety

Blood samples for hematology are collected in 4 ml EDTA tubes and sent to the hematology laboratory of the Leiden University Medical Center (LUMC) for routine analysis. Blood samples for blood chemistry are collected in 4 ml Heparin tubes and sent to the clinical chemistry laboratory for routine analysis.

Drug Concentration Measurements

Frozen serum samples for PK analysis are stored in the freezer at −40° C. and are shipped on dry ice to the laboratory, at the end of the study. Samples are analyzed using a validated LC-MS method for the determination of testosterone levels. It is not possible to discriminate endogenous and exogenous testosterone from each other using this method.

Quality Assurance

The study is conducted in compliance with the pertaining CHDR Standard Operating Procedures and CHDR's QA procedures.

Calculation of Pharmacokinetic Parameters

A validated LC-MS/MS method is employed to determine serum testosterone. All samples from study participant completing both the periods are analyzed.

Incurred sample reanalysis is performed:

-   -   C_(min), C_(max), and t_(max) actual measured values. Values are         determined relative to the testosterone administration time in         treated subjects.     -   Area under the concentration curve (AUC) is estimated for the 0         to 12 hour time interval using the trapezoidal rule.     -   Significance is evaluated using the t-test. Additional         exploratory analyses of PK parameters could be performed as         necessary.

The relative pharmacokinetic profile of the pre-filled syringe and the multiple dose dispenser is determined using the AUC_(0-12h) and Cmax_(0-12h) corrected for the endogenous serum testosterone concentration. For bioequivalence, the relative mean of the dispenser to the pre-filled syringe using log transformed data for AUC_(0-12h) and Cmax_(0-12h) is corrected for the endogenous serum testosterone concentration, is determined to be between 80% to 125%.

Analysis of Safety Parameters

The Day 5 close-out findings is compared to the screening results and clinically significant changes were to be identified in the following:

-   -   1. Vital Signs and Adverse Events: Blood Pressure, Body         Temperature, Respiratory Rate, Heart Rate.     -   2. Otorhinolaryngological examination with the nasal tolerance         data presented in summary tables.     -   3. Complete Blood Count: white blood count, hemoglobin and         hematocrit.     -   4. Clinical chemistry profile: sodium, potassium, chloride,         glucose, urea, creatinine, calcium, phosphate, uric acid, total         bilirubin, albumin, AST, ALT, ALP, GGT, CK and cholesterol.     -   5. Urinalysis.

Determination of Sample Size

As this is a relatively small Phase I PK study with the intent to compare a pharmacokinetic profile of testosterone after administration of TBS-1 from two different dispensers in healthy male subjects, a true sample size calculation is not performed.

Subjects

26 Subjects are enlisted

-   -   2 subjects are not screened due to planning problems     -   1 subject is not screened because he does not have a general         practitioner

23 Subjects are screened

-   -   3 screening failures due to ENT abnormalities     -   1 screening failure due to positive hepatitis B test     -   1 screening failure due to positive hepatitis C test

18 Subjects passed screening

-   -   12 subjects are randomized and completed the study     -   1 subject is cancelled before the baseline visit due to         concurrent illness     -   5 subjects are reserves, but not needed

No subjects discontinue after randomization.

Efficacy Evaluation

Data collected is used in the analysis. This yields three PK curves of 12 hours each, one without treatment (baseline), and one each after administration of TBS-1 using the multiple dose dispenser or syringe.

Demographic Characteristics

Subject demographics are summarized in Table 4 below.

TABLE 4 Subject demographics Variable N MEAN STD MIN MAX Age (yrs) 12 23.4 3.0 18 28 BMI (kg/m²) 12 23.55 2.45 20.9 28.4 Height (cm) 12 184.43 8.46 173.5 197.0 Weight (kg) 12 80.08 9.76 63.2 98.2

Measurements of Treatment Compliance

The nasal gel is self-administered by subjects. All administrations are successful.

Efficacy Results and Tabulations of Individual Patient Data

FIG. 24 shows the individual serum testosterone levels per occasion (baseline without medication, TBS-1 using the multiple dose dispenser and TBS-1 using syringes), where T=0 occurred at 21:00 hours clock time. FIG. 24 shows the individual and median testosterone concentration versus time grouped by treatment.

All subjects have testosterone levels within the normal range (24 hour C_(mean)≥300 ng/dL and ≤1050 ng/dL). The baseline curves clearly show the slow circadian fluctuations in testosterone levels that are expected in a young, healthy population with the highest levels in the early morning.

Although dose and volume of TBS-1 that is administered is exactly the same for both forms of administration, the graphs in FIGS. 15 and 16 suggest that there are differences in pharmacokinetic profile.

Pharmacokinetic Parameters

The following primary pharmacokinetic parameters, per occasion, are calculated:

-   -   AUC₀₋₁₂: Area under the serum concentration-time curve         (ng·hr/dL) for each occasion from 21:00 to 9:00 hrs, is         calculated using the linear trapezoidal method.     -   C_(mean): Mean concentration (ng/dL) during each occasion from         21:00 to 9:00 hrs, is calculated as AUC_0-12/12.     -   C_(max): Maximum is observed concentration (ng/dL) during each         occasion.     -   C_(min): Minimum is observed concentration (ng/dL) during each         occasion.     -   t_(max): Time (hr) at which C_(max) is observed.

Tables 5 to 7 below summarize the primary pharmacokinetic parameters for endogenous testosterone during the baseline visit when no treatment is administered, for TBS-1 when administered using the multiple dose dispenser, and for TBS-1 when administered using a syringe.

Testosterone, Baseline, No Treatment

TABLE 5 Testosterone, no treatment Parameter Mean SD Median Min Max N AUC₀₋₁₂ 4911 1156 4726 3337 7164 12 t_(max) 8.833 3.486 10.0 2.0 12 12 C_(max) 514.2 117.5 480.0 384.0 746 12 C_(min) 298.6 89.01 308.0 134.0 453 12 C_(mean) 409.0 96.4 392.8 278.1 597 12 AUC₀₋₁₂ in ng*hr/dL; t_(max) in hours; C_(max), C_(min) and C_(mean) in ng/dL

Testosterone, TBS-1 Multiple Dose Dispenser

TABLE 6 Testosterone, TBS-1 multiple dose dispenser Parameter Mean SD Median Min Max N AUC₀₋₁₂ 7484 1798 7347 4847 11350 12 t_(max) 2.751 3.961 1.25 0.3333 12 12 C_(max) 1028 283.1 970.5 645 1440 12 C_(min) 337.9 119.7 328.5 145 565 12 C_(mean) 623.6 149.9 612.3 403.9 945.7 12 AUC₀₋₁₂ in ng*hr/dL; t_(max) in hours; C_(max), C_(min) and C_(mean) in ng/dL

Testosterone, TBS-1 Syringe

TABLE 7 Testosterone, TBS-1 syringe Parameter Mean SD Median Min Max N AUC₀₋₁₂ 7266 1360 7237 5186 9371 12 t_(max) 5.612 4.736 5.0 0.667 12 12 C_(max) 778.8 144.1 754.5 543 1100 12 C_(min) 355.9 66.96 337.0 291 498 12 C_(mean) 605.4 113.2 603.1 432.2 780.9 12 AUC₀₋₁₂ in ng*hr/dL; t_(max) in hours; C_(max), C_(min) and C_(mean) in ng/dL

The listing of individual primary pharmacokinetic parameters is included in Table 7A.

TABLE 7A Efficacy Data Individual PK Parameters Individual PK parameters 0-12 hrs for each occasion Subject Occasion Treatment AUC_0-12t_max C_max C_mean C_min 1 1 No Treatment 5722 10.0000 600 476.9 321 1 2 TBS-1 mdd 9394 12.0000 1070 782.9 340 1 3 TBS-1 syringe 7802 12.0000 840 650.1 400 2 1 No Treatment 3731 10.0000 388 310.9 242 2 2 TBS-1 syringe 7367 1.5000 779 613.9 333 2 3 TBS-1 mdd 7592 0.3333 1420 632.7 386 3 1 No Treatment 4771 3.0000 498 395.4 332 3 2 TBS-1 mdd 6056 0.6667 645 504.7 395 3 3 TBS-1 syringe 7107 5.0000 691 592.3 312 4 1 No Treatment 7164 2.0000 746 597.0 453 4 2 TBS-1 syringe 8639 6.0000 837 720.0 498 4 3 TBS-1 mdd 8370 0.3333 1440 697.5 500 5 1 No Treatment 3337 10.0000 384 278.1 134 5 2 TBS-1 mdd 4847 0.3500 1280 403.9 145 5 3 TBS-1 syringe 5439 1.0000 725 453.3 292 6 1 No Treatment 3673 10.0000 422 305.2 166 6 2 TBS-1 syringe 5186 10.0200 543 432.2 304 6 3 TBS-1 mdd 5851 1.0000 715 487.6 325 7 1 No Treatment 4681 12.0000 456 390.1 324 7 2 TBS-1 syringe 6250 12.0000 661 520.8 291 7 3 TBS-1 mdd 6503 1.5000 881 541.2 159 8 1 No Treatment 4632 12.0000 473 386.0 295 8 2 TBS-1 mdd 7102 1.5000 813 591.9 332 8 3 TBS-1 syringe 8529 0.6667 1100 710.7 343 9 1 No Treatment 4222 12.0000 481 351.8 287 9 2 TBS-1 mdd 11350  3.0000 1350 945.7 276 9 3 TBS-1 syringe 6992 12.0000 730 582.7 341 10 1 No Treatment 6503 10.0000 718 541.9 397 10 2 TBS-1 syringe 9371 5.0000 874 780.9 445 10 3 TBS-1 mdd 8747 10.0000 820 728.9 565 11 1 No Treatment 5541 5.0000 525 461.7 353 11 2 TBS-1 mdd 7823 2.0000 848 651.9 315 11 3 TBS-1 syringe 8550 1.5000 898 710.6 408 12 1 No Treatment 4950 10.0000 479 412.5 279 12 2 TBS-1 syringe 5962 0.6667 668 496.8 304 12 3 TBS-1 mdd 6171 0.3333 1060 514.2 317 mdd - multiple dose dispenser

Total testosterone exposure is estimated by the mean area under the serum concentration-time curve (AUC₀₋₁₂ in ng·hr/dL) is higher after TBS-1 administration using the dispenser or syringe than endogenous levels alone (7484 and 7266, respectively, versus 4911 ng*h/dL). Between the methods of administration, the difference in mean AUC₀₋₁₂ is small. The significance of this difference is explored below.

Unexpectedly, mean C_(max) is higher after administration with the dispenser than when with a syringe (1028 versus 778.8 ng/dL, respectively). T_(max) occurs sooner after administration using the dispenser than after the syringe (2.75 versus 5.6 hours, respectively). Thus, after administration using the multiple dose dispenser serum testosterone seems to be absorbed faster than with a syringe. The significance of these differences is explored below.

Two subjects reach t_(max) of testosterone only 10 and 12 hours after administration with the dispenser. In three subjects, t_(max) is 10 and 12 hours after administration with the syringe, and t_(max) is 5 and 6 hours in two others. Most likely, the endogenous testosterone peak fluctuation exceeded levels that is caused by exogenous testosterone administration. Thus, the calculated mean t_(max) may be faster when testosterone is dosed high enough that the peak caused by exogenous administration exceeds the endogenous peak.

Derived Pharmacokinetic Parameters

The following derived pharmacokinetic parameters, combining results from occasions, are calculated:

-   -   AUC_(0-12_drug): difference between AUC₀₋₁₂ after treatment         (syringe or dispenser) and no treatment (baseline occasion)     -   C_(max_drug): difference between C_(max) after treatment         (syringe or dispenser) and the observed concentration at t_(max)         in absence of treatment (baseline occasion)     -   Ratio AUC_(0-12_drug): % ratio between AUC_(0-12_drug) using         dispenser and syringe     -   Ratio C_(max_drug): % ratio between C_(max_drug) using dispenser         and syringe     -   Mean and uncertainty (95%, 90% and 80% confidence interval) of         the log of Ratio AUC_(0-12_drug)     -   Mean and uncertainty (95%, 90% and 80% confidence interval) of         the log of Ratio C_(max_drug)

Testosterone Level Using TBS-1, Baseline Subtracted

Tables 8 and 9 below show the AUC and C_(max) for the different TBS-1 delivery methods after subtracting baseline levels of testosterone.

TABLE 8 Testosterone level using TBS-1 multiple dose dispenser, baseline subtracted Parameter Mean SD Median Min Max N AUC₀₋₁₂_drug 2573.0 1679.0 2211 1207 7126 12 C_(max)_drug  630.8  314.7  534  102 1111 12

TABLE 9 Testosterone level TBS-1 syringe, baseline subtracted Parameter Mean SD Median Min Max N AUC₀₋₁₂_drug 2356.0 900.9 2219 1012 3897 12 C_(max)_drug  379.9 177.1  357  121  782 12

Testosterone Level TBS-1 Dispenser Over Syringe Ratio

Table 10 below shows the ratio of serum testosterone levels that are reached with the dispenser or syringe, after subtracting baseline testosterone levels. There is clearly a difference in C_(max) between the administration forms (mean ratio dispenser over syringe C_(max) 2.057), but the AUCs are comparable (mean ratio dispenser over syringe AUC 1.12).

TABLE 10 Testosterone, ratio of TBS-1 multiple dose dispenser over syringe Parameter Mean SD Median Min Max N Ratio AUC₀₋₁₂_drug 1.122 0.580 0.940 0.550 2.572 12 Ratio C_(max)_drug 2.057 1.339 1.983 0.227 4.455 12 logRatio AUC₀₋₁₂_drug 0.014 0.453 −0.071 −0.598 0.945 12 logRatio C_(max)_drug 0.455 0.860 0.684 −1.484 1.494 12

Table 11 below shows the log of the ratio of serum testosterone levels that are reached when administering using the multiple dose dispenser over syringe, after subtracting baseline testosterone levels, with 95%, 90% and 80% confidence intervals.

When plotting probability density of the log ratio of testosterone levels that are reached with the multiple dose dispenser over levels that are reached with the syringe as shown in FIG. 17, no significant difference is demonstrated for either AUC₀₋₁₂ or C_(max) within 95% confidence intervals. There is a trend toward a difference for C_(max). However, this data does not confirm bioequivalence at a confidence interval level of 90% for either AUC₀₋₁₂ or C_(max), as the study is not powered for 2-one-sided tests.

TABLE 11 Testosterone TBS-1 log ratios with different confidence intervals Parameter Mean CI (%) LLCI ULCI logRatio 0.01398 95 −0.27400 0.3019 AUC₀₋₁₂_drug 90 −0.2209574 0.24892 80 −0.16438 0.19234 logRatio 0.45520 95 −0.09145 1.0020 C_(max)_drug 90 0.00917 0.90127 80 0.11658 0.79386 CI = confidence interval; log(0.8) = −0.22314; log(1.25) = 0.22314

Handling of Dropouts or Missing Data

No subjects drop out of the study. Blinded data review did not lead to removal of any data points.

Extent of Exposure

The pharmacokinetic results show that exposure to testosterone is only higher than the upper level of the normal range very briefly shortly after TBS-1 administration.

Adverse Events (AEs)

Treatment is well tolerated. There are 12 adverse event reports in total. Three events had their onset before the first administration of study medication and are therefore unrelated. Four reports of mild complaints such as sore throat are considered unlikely to be caused by study medication when considering the nature of the complaints and the time lapse after administration. One subject reschedules one occasion because of gastro-intestinal complaints that are unlikely to be related to study medication, onset of symptoms is days after study drug administration. Symptoms resolve without treatment.

Reports of bad smell and taste are the only complaints that are considered clearly related to administration of medication. These complaints are mild in intensity and could be considered a product characteristic rather than a medical condition. Bad smell and taste complaints do not lead to discontinuation of the study medication and diminishes with repeated dosing.

Display of Adverse Events

A listing of adverse events is included in Table 12.

TABLE 12 Listing of Adverse Events Treatment Treatment action Start Diagnosis SAE related Subject Visit Symptoms Chronicity Duration Severity TBS-1 mdd  2 3 6 APR. 2011 8:30 OROPHARYNGEAL PAIN None Irritated single occasion 0D01H20M mild No unlikely throat.  3 2 30 MAR. 2011 12:00 HEADACHE None Headache single occasion 0D09H00M mild No unrelated 30 MAR. 2011 21:04 APPLICATION SITE ODOUR None Smells nasty, single occasion 0D02H55M mild No definitely bad taste.  5 2 30 MAR. 2011 20:40 APPLICATION SITE ODOUR None It smells nasty. single occasion 0D00H30M mild No definitely 30 MAR. 2011 21:15 DYSGEUSIA None Bad taste. single occasion 0D00H45M mild No definitely  8 2 13 APR. 2011 20:45 CATHETER SITE RASH Removed plastic Red rash in single occasion 1D18H15M mild No unrelated tape patch. left armpit, where cannula is placed. TBS-1 syringe  1 3 6 APR. 2011 8:30 OROPHARYNGEAL PAIN None Sore throat, single occasion 0D00H40M mild No unlikely  2 2 31 MAR. 2011 13:00 AGITATION None Feeling single occasion 0D20H00M mild No unlikely agitated.  4 2 30 MAR. 2011 20:45 APPLICATION SITE ODOUR None It smells nasty. single occasion 0D00H20M mild No definitely  6 2 30 MAR. 2011 20:33 APPLICATION SITE ODOUR None It smells nasty. single occasion 0D00H27M mild No definitely 10 2 18 APR. 2011 23:00 DIARRHOEA None Nausea, single occasion 1D21H00M mild No unlikely diarrhoea. No Treatment 11 1 13 APR. 2011 9:19 HEADACHE Paracetamol, Headache single occasion 0D06H41M mild No unrelated sleep. Note: mdd = multiple dose dispnenser M = Missing U = Unknown

Analysis of Adverse Events

All adverse events are considered mild and are transient. Nasal tolerance is good. Initial complaints of bad smell or taste did not lead to discontinuation of the study.

Deaths, Other Serious Adverse Events, and Other Significant Adverse Events

There are no deaths, serious adverse events or other significant adverse events.

Evaluation of Each Laboratory Parameter

There are no abnormal hematology, blood chemistry or urine laboratory findings that are considered clinically significant in the opinion of the investigator.

Vital Signs, Physical Findings and Other Observations Related to Safety

There are no abnormal findings in vital signs, on physical examinations or other observations that are considered clinically significant in the opinion of the investigator.

Safety Conclusions

Treatment is well tolerated, nasal tolerance is good. All adverse events are considered mild and are transient. Initial complaints of bad smell or taste did not lead to study discontinuation.

Discussion and Overall Conclusions

This study compares the pharmacokinetic profile of TBS-1 testosterone nasal gel administered using a multiple dose dispenser to the profile of TBS-1 delivery using a syringe. In order to avoid carry-over effects that are caused by repeated dosing, the order of administration is randomized. Prior to first administration, subjects are admitted to the unit for blood sampling in order to determine a baseline testosterone profile.

All 12 subjects, age range 18 to 28 years, complete the study successfully. Although not assessed at screening, all subjects have baseline testosterone levels within the normal range. Treatment is well tolerated and all reported adverse events are transient and considered mild. Complaints of bad smell and taste are reported, although this did not lead to discontinuation and decreased with repeated dosing.

As expected, the total exposure to testosterone (as estimated by the mean area under the serum concentration-time curve (AUC₀₋₁₂)) after TBS-1 administration using the dispenser or syringe exceed endogenous levels. The difference in mean AUC₀₋₁₂ between the two modes of administration is small.

Unexpectedly, mean C_(max) is considerably higher after administration with the dispenser than when administering using a syringe. T_(max) is also earlier after administration using the dispenser than after the using the syringe. Thus, testosterone absorption seems to be faster with the multiple dose dispenser than with a syringe, but the total absorbed amount is similar.

Two subjects reach t_(max) of testosterone only 10 and 12 hours after administration with the dispenser. In three subjects, t_(max) is 10 and 12 hours after the syringe, and t_(max) is 5 and 6 hours in two others. Most likely, the endogenous testosterone peak fluctuation exceed levels that are caused by exogenous testosterone administration. Thus, the calculated mean t_(max) may be faster when testosterone is dosed high enough that the peak caused by exogenous administration exceeds the endogenous peak.

When plotting probability density of the log ratio of testosterone levels that are reached with the multiple dose dispenser over levels that are reached with the syringe, no significant difference is demonstrated for either AUC₀₋₁₂ or C_(max) within 95% confidence intervals. There is a trend toward a difference for C_(max). However, this data does not confirm bioequivalence at a confidence interval level of 90% for either AUC₀₋₁₂ or C_(max). This finding may be due to the fact that the ideal positioning of the delivering tip is easier to find with the multiple dose device than the syringe.

Also, in accordance with this Example 6, see FIGS. 23 and 24.

The following formulations are in Table 13 used in Examples 5-7 and in FIGS. 23 and 24.

TABLE 13 TBS1V TBS-1A 4% TBS-1A 4% Material TBS1 (vs. H20) (A) alternate (B) TBS-1A 8% Dimethyl isosorbide 0 0 25.0 15.0 25.0 Diethyleneglycol ethyl ether 0 0 10.0 5.0 10.0 Povidone 0 0 3.0 3.0 3.0 Copovidone 0 0 2.0 2.0 2.0 Hydroxypropyl cellulose 0 0 0.5 0.5 0.5 Testosterone micronized 4.0 4.0 4.0 4.0 8.0 Castor oil 88.0 87.95 50.5 65.5 46.5 Labrafil M1944CS 4.0 4.0 0 0 0 Colloidal silicon dioxide 4.0 4.0 5.0 5.0 5.0 Water 0 0.05 0 0 0 Total 100.0 100.0 100.0 100.0 100.0

Example 13 A Phase 3, 90-Day, Randomized, Dose-Ranging Study, Including Potential Dose Titration, Evaluating the Efficacy and Safety of Intranasal TBS-1 in the Treatment of Male Hypogonadism with Sequential Safety Extension Periods of 90 and 180 Days Investigational Product: 4.5% TBS-1 Intranasal Testosterone Gel Protocol Number: TBS-1-2011-03 Synopsis TITLE: A 90-Day, Randomized, Dose-Ranging Study, Including Potential Dose Titration, Evaluating the Efficacy and Safety of Intranasal TBS-1 in the Treatment of Male Hypogonadism With Sequential Safety Extension Periods of 90 and 180 Days PROTOCOL NUMBER: TBS-1-2011-03

INVESTIGATIONAL PRODUCT: TBS-1 intranasal 4.5% testosterone gel

PHASE: 3

INDICATION: Adult male hypogonadism (primary and secondary)

OBJECTIVES:

The primary objective of the study is to determine the efficacy of 4.5% TBS-1 gel, administered as 2 or 3 daily intranasal doses of 5.5 mg per nostril, as demonstrated by an increase in the 24-hour average concentration (C_(avg)) of serum total testosterone to the normal range (≥300 ng/dL and ≤1050 ng/dL) in ≥75% of male subjects treated for hypogonadism. See also Exhibit C (the contents of which are incorporated herein by reference).

The secondary objectives of this study are the following:

-   -   To determine the efficacy of 4.5% TBS-1 gel, administered 2 or 3         times daily at a dose of 5.5 mg per nostril, in achieving the         following for serum total testosterone maximum concentration         (C_(max)):         -   C_(max)≤1500 ng/dL in ≥85% of subjects,         -   C_(max) 1800 to 2500 in <5% of subjects, and         -   C_(max)>2500 ng/dL in no subjects;     -   To determine the safety and tolerability of TBS-1 after 90, 180,         and 360 days of treatment;     -   To determine the effect of TBS-1 treatment on body composition         (total body mass, lean body mass, fat mass, and percent fat);     -   To determine the effect of TBS-1 treatment on bone mineral         density (lumbar spine and hip);     -   To determine the effect of TBS-1 treatment on mood;     -   To determine the effect of TBS-1 treatment on erectile function;         and

To determine the serum concentration and pharmacokinetics (PK) of total testosterone, dihydrotestosterone (DHT), and estradiol after TBS-1 administration.

Population:

The population for this study is adult men 18 to 80 years of age, inclusive with fasting morning (0900 h±30 min) total serum testosterone <300 ng/dL. Subjects currently treated with testosterone must undergo 2 to 4 weeks of washout depending on the route of administration.

Study Design and Duration:

This is a Phase 3, 2-group, multicenter study consisting of 4 study periods including 2 safety extension periods as follows:

-   -   A 3- to 7-week Screening Period that includes medication washout         for subjects currently receiving testosterone treatment;     -   A 90-day randomized, open-label Treatment Period during which         subjects will receive 5.5 mg per nostril of 4.5% TBS-1 twice         daily (BID) or three times daily (TID) with potential daily dose         adjustment on Day 45 for subjects in the BID treatment group as         determined by the serum total testosterone PK profile;     -   A 90-day open-label Safety Extension Period (Safety Extension         Period 1) for all study subjects; and     -   An additional 180-day open-label Safety Extension Period (Safety         Extension Period 2) for a subset of 75 subjects.

The approximate total duration of study participation for subjects completing all 4 periods will be up to 406 days (˜58 weeks).

Screening Period

The Screening Period will take place over 3 to 7 weeks and will consist of up to 3 study visits. The duration of screening will depend on whether subjects are naïve to testosterone treatment or if they are currently being treated with a testosterone product. Subjects currently being treated with a testosterone product will require a washout. The duration of washout will depend on the type of testosterone therapy and the date of their last dose. For subjects taking testosterone injections, there must be at least 4 weeks between their last testosterone injection and the first measurement of morning serum total testosterone for qualification. For subjects taking oral, topical, or buccal testosterone, there must be at least 2 weeks between the last administration of testosterone and the first measurement of morning serum total testosterone for qualification.

Visit 1 will occur up to 7 weeks (Week −7) prior to randomization for subjects currently receiving testosterone injections, up to 5 weeks (Week −5) prior to randomization for subjects currently receiving oral, topical, or buccal testosterone, and up to 3 weeks (Week −3) prior to randomization for naïve subjects. During Visit 1, informed consent will be obtained and the subject's inclusion and exclusion criteria will be assessed based on medical interview, concomitant medications, physical examination, digital rectal examination (DRE) of the prostate, vital sign measurements, and screening laboratory evaluations. For naïve subjects, a fasting morning (0900 h±30 min) serum total testosterone level and baseline laboratory measurements will be assessed at Visit 1.

Non-naïve subjects will be instructed to discontinue all testosterone therapies at Visit 1. After Visit 1, if it is determined that a subject does not qualify for the study, the subject will be notified and instructed to restart prior testosterone therapy.

Subjects undergoing washout from testosterone therapy will return for Visit 1.1 and will have fasting morning (0900 h±30 min) serum total testosterone levels and baseline laboratory measurements obtained. For subjects undergoing washout of testosterone injections, Visit 1.1 will occur 4 weeks after the last testosterone injection (up to Week −3). For subjects undergoing washout of oral, topical, or buccal testosterone, Visit 1.1 will occur 2 weeks after the last administration of testosterone (up to Week −3). Visit 1.1 is not required for naïve subjects.

At Visit 2 (up to Week −2), all subjects will have a fasting morning (0900 h±30 min) serum total testosterone level and 12-lead electrocardiogram (ECG) assessed. At the screening visits (Visits 1, 1.1, and 2), serum total testosterone levels will be measured using a validated assay developed by Medpace Reference Laboratories. The results will be used for determination of a subject's inclusion or exclusion from the study. To be included in the study, subjects must have 2 fasting morning (0900 h±30 min) serum total testosterone levels <300 ng/dL. In subjects with a known history of male hypogonadism, if 1 of the 2 serum total testosterone levels is 300 ng/dL, the serum total testosterone level may be retested once. After retesting, if 2 of the 3 levels are <300 ng/dL, then the subject will be eligible to participate in the study.

Subjects who qualify for the study based on screening assessments at Visits 1, 1.1, and 2 will be scheduled for an otorhinolaryngological (ENT) examination with nasal endoscopy performed by an ENT specialist. All qualified subjects will also have dual-energy x-ray absorptiometry scans scheduled in the interval between Visit 2 and randomization (Visit 3) for the assessment of body composition and bone mineral density.

Treatment Period

The randomized, open-label Treatment Period will consist of 4 study visits: Visit 3 (Day 1), Visit 4 (Day 30), Visit 5 (Day 60), and Visit 6 (Day 90).

Visit 3 (Day 1) will take place in the evening. At Visit 3, subjects will be randomized in a 3:1 ratio to 1 of the following 2 treatment groups:

-   -   5.5 mg per nostril of 4.5% TBS-1 BID or     -   5.5 mg per nostril of 4.5% TBS-1 TID.

Baseline levels of fasting serum total testosterone, DHT, and estradiol will be measured. Study drug (TBS-1) will be administered at 2100 h and 0700 h in the BID treatment group (total daily dose of 22 mg/day) and at 2100 h, 0700 h, and 1300 h in the TID treatment group (total daily dose of 33 mg/day). The first dose of study drug will be administered at Visit 3 (Day 1) at 2100 h and training on drug administration will be provided to subjects. Subjects will be asked to maintain a daily diary documenting administration of study drug doses throughout the Treatment Period, Safety Extension Period 1, and Safety Extension Period 2.

At Visit 4 (Day 30 to Day 31), study drug will be administered at the site, beginning with the 2100 h dose of TBS-1. Subjects will be required to remain at the site for 24 hours after the 2100 h drug administration and complete post-dose PK profiles for serum total testosterone, DHT, and estradiol will be obtained. The 24-hour C_(avg) of serum total testosterone for subjects in the BID group will be estimated based on the sum of serum total testosterone levels collected at 2 sampling points during the 24-hour PK profile: the sample collected at 9.0 hours (at 1 hour before the morning 0700 h dose) and the sample collected at 10.33 hours (20 minutes after the morning 0700 h dose). The following titration criteria will be used:

-   -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is <755 ng/dL,         then the estimated 24-hour C_(avg) is <300 ng/dL and     -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is ≥755 ng/dL,         then the estimated 24-hour C_(avg) is ≥300 ng/dL.

Subjects randomized to the BID group with an estimated serum total testosterone C_(avg)<300 ng/dL, will be contacted by phone and instructed to increase the daily dose of TBS-1 to TID on Day 45. The decision to increase the subject's daily dose to TID will be made by the investigator based on the criteria specified above. This daily dose will be continued throughout the remainder of the Treatment Period and, as applicable, both Safety Extension Periods.

At Visit 6 (Day 90 to Day 91), study drug will be administered at the site, beginning with the 2100 h dose of TBS-1. Subjects will be required to remain at the site for 24 hours after the 2100 h drug administration and complete post-dose PK profiles for serum total testosterone, DHT, and estradiol will be obtained.

At Visits 3, 4, and 6, serum total testosterone, DHT, and estradiol levels will be measured using a sensitive and specific assay developed and validated by Analytisch Biochemisch Laboratorium BV. The results will be used for PK analyses.

Safety Extension Period 1

All subjects will continue into Safety Extension Period 1 and will be instructed to continue their current daily dose of TBS-1 for the 90-day Safety Extension Period (Day 90 to Day 180). Subjects will return to the site for monthly visits.

Safety Extension Period 2

A subset of approximately 75 subjects will continue in the study for an additional 180-day Safety Extension Period (Day 180 to Day 360). The subset of subjects who continue into Safety Extension Period 2 will consist of the first subjects to complete Safety Extension Period 1. For the duration of Safety Extension Period 2, subjects will remain on the same daily dose of TBS-1 administered on Day 90 of the Treatment Period and throughout Safety Extension Period 1. Subjects will return to the site for monthly visits.

Dosage Forms and Route of Administration:

Study Drug: 4.5% TBS-1 Pharmaceutical form: Gel for intranasal administration Content: Active ingredient: testosterone Excipients: silicon dioxide, castor oil, and oleoyl polyoxylglycerides Mode of Intranasal administration: Batch number: To be determined Storage conditions: Between 15-25° C.

TBS-1 is administered intranasally by the subject. A multiple-dose dispenser will be used for gel deposition into the nasal cavity. The dispenser is a finger-actuated dispensing system designed to deliver 5.5 mg of 4.5% TBS-1 gel per actuation from a non-pressurized container into the nasal cavity. The dispenser is designed to administer 45 doses (90 actuations) of TBS-1. The key components of the multiple-dose dispenser include a barrel, base, pump, and actuator, which are composed of polypropylene, and a piston, which is composed of polyethylene.

Efficacy Variables:

The primary efficacy variable is the number and percentage of subjects with a serum total testosterone C_(avg) value within the normal range (≥300 ng/dL and ≤1050 ng/dL) on Day 90.

Secondary efficacy variables include the following:

-   -   The number and percentage of subjects with a serum total         testosterone maximum concentration (C_(max)) value in the         following ranges on Day 90:         -   ≤1500 ng/dL,         -   >1500 and <2500 ng/dL, and         -   ≥2500 ng/dL;     -   The number and percentage of subjects with a serum total         testosterone C_(avg) value in the normal range (≥300 ng/dL and         ≤1050 ng/dL) on Day 30;     -   The number and percentage of subjects with a serum total         testosterone C_(max) value in the following ranges on Day 30:         -   ≤1500 ng/dL,         -   >1500 and <2500 ng/dL, and         -   ≥2500 ng/dL;     -   The complete PK profile (including C_(avg), the minimum         concentration, C_(max), and time to maximum concentration) of         serum total testosterone on Day 30 and Day 90;     -   The time within the normal range for serum total testosterone         based on the PK profile on Day 30 and Day 90;     -   The PK profile of serum estradiol on Day 30 and Day 90;     -   The PK profile of serum DHT on Day 30 and Day 90;     -   The ratio of DHT C_(avg) to total testosterone C_(avg) on Day 30         and Day 90;     -   The Positive and Negative Affect Schedule scores at baseline,         Day 30, Day 60, and Day 90;     -   The International Index of Erectile Function scores at baseline,         Day 30, Day 60, and Day 90;     -   Change in bone mineral density from baseline to Day 180; and

Change in body composition (total body mass, lean body mass, fat mass, and percent fat) from baseline to Day 180.

Safety Variables:

Safety assessments will include adverse events, clinical laboratory measurements (chemistry profile, liver function tests, fasting lipid profile, hematology, urinalysis, glycosylated hemoglobin, prostate specific antigen, and endocrine profile), 12-lead ECG parameters, vital signs (blood pressure, heart rate, temperature, and respiratory rate), physical examination parameters, DREs of the prostate, and ENT examinations.

Statistical Analyses:

The intent-to-treat (ITT) population will consist of all subjects who receive randomized study drug and have at least 1 valid post-baseline efficacy measurement. The safety population will consist of all subjects who receive randomized study drug and have safety measurements during the treated periods. The efficacy analyses will be based on the ITT population and the safety analyses will be based on the safety population. The primary efficacy parameter, the C_(avg) of serum total testosterone at Day 90, will be calculated from the area under the curve (AUC) using the following formula:

C _(avg)=AUC_(0-24h)/24

The AUC curve for both BID and TID dosing regimens will be determined for the 0 to 24-hour time interval by using the linear trapezoidal rule.

The number and percentage of subjects who reach the treatment goal (ie, serum total testosterone C_(avg) value in the normal range) at Day 90 or Early Termination will be summarized descriptively. The analysis and calculation for the frequency of attaining the secondary study objectives will be performed using similar methods.

The concentrations of serum total testosterone, DHT, and estradiol will be provided for baseline, Day 90 or Early Termination, and the change from baseline to Day 90 or Early Termination.

The same summary will be performed at Day 30 for the purpose of comparing the treatment difference between BID and TID after 30 days of treatment.

For other efficacy measurements, descriptive statistics will be provided at each visit. If appropriate, the change from baseline to post-baseline visits will be determined. The descriptive summary will also be provided for the safety extension periods.

In addition, the Day 30 24-hour C_(avg) serum total testosterone values for all subjects in the BID treatment group will be compared to the estimated value determined by the titration criteria. The acceptability of the titration criteria will be assessed.

Adverse events will be coded using the latest version of the Medical Dictionary for Regulatory Activities. A general summary of the adverse events and serious adverse events for each treatment group will be presented by the overall number of adverse events, the severity, and the relationship to study drug. The incidence of adverse events will be summarized by system organ class, preferred term, and treatment group. The safety laboratory data will be summarized by visit and by treatment group along with the change or percent change from baseline. Vital signs will also be summarized by visit and by treatment group along with the change from baseline. The clinical findings in the physical examination and 12-lead ECG results will be summarized at each scheduled visit. Other safety measurements will be summarized and listed if deemed necessary.

Sample Size Determination:

A sample size of approximately 280 subjects (210 subjects randomized to the BID treatment group and 70 subjects randomized to the TID treatment group) was selected to provide a sufficient number of subjects to determine the efficacy, safety, and tolerability of intranasal 4.5% TBS-1 gel. Since this is an observational study, no formal sample size calculation was performed.

Preliminary data on 139 hypogonadal men who have completed 30 days of BID or TID treatment of the Phase 3 Study exhibit the following results, established by in accordance with the titration methods set forth in Example 15 below and as described herein:

-   -   107 males were treated with the BID dosing regimen, 4.5% TBS-1,         and 32 males on the TID regimen     -   Approximately 80% of the males treated with 4.5% TBS-1 achieved         an average testosterone level above 300 ng/dl     -   Both the BID and TID treatment groups had more than 75% of the         patients above the average testosterone level 300 ng/dl cut-off.

In accordance with the present invention, an exemplary label is provided in Exhibit E (the contents of which are incorporated herein by reference).

Example 14 Statistical Analysis Plan A 90-Day, Randomized, Dose-Ranging Study, Including Potential Dose Titration, Evaluating the Efficacy and Safety of Intranasal TBS-1 in the Treatment of Male Hypogonadism with Sequential Safety Extension Periods of 90 and 180 Days Investigational Product: 4.5% TBS-1 Intranasal Testosterone Gel Protocol Number: TBS-1-2011-03 Introduction

This example provides a description of the statistical methods and procedures to be implemented for the analyses of data from the study with protocol number TBS-1-2011-03. See also Exhibit C (the contents of which are incorporated herein by reference).

Study Design and Objectives Study Objectives Primary Objective

The primary objective of the study is to determine the efficacy of 4.5% TBS-1 gel, administered as 2 or 3 daily intranasal doses of 5.5 mg per nostril, as demonstrated by an increase in the 24-hour average concentration (C_(avg)) of serum total testosterone to the normal range (≥300 ng/dL and ≤1050 ng/dL) in 75% of male subjects treated for hypogonadism.

Secondary Objective

The secondary objectives of this study are the following:

-   -   To determine the efficacy of 4.5% TBS-1 gel, administered 2 or 3         times daily at a dose of 5.5 mg per nostril, in achieving the         following for serum total testosterone maximum concentration         (C_(max)):         -   C_(max)≤1500 ng/dL in ≥85% of subjects,         -   C_(max) 1800 to 2500 in <5% of subjects, and         -   C_(max)>2500 ng/dL in no subjects;     -   To determine the safety and tolerability of TBS-1 after 90, 180,         and 360 days of treatment;     -   To determine the effect of TBS-1 treatment on body composition         (total body mass, lean body mass, fat mass, and percent fat);     -   To determine the effect of TBS-1 treatment on bone mineral         density (lumbar spine and hip);     -   To determine the effect of TBS-1 treatment on mood;     -   To determine the effect of TBS-1 treatment on erectile function;         and     -   To determine the serum concentration and pharmacokinetics (PK)         of total testosterone, dihydrotestosterone (DHT), and estradiol         after TBS-1 administration.

Study Design and Duration

This is a Phase 3, 2-group, multicenter study consisting of 4 study periods including 2 safety extension periods as follows:

-   -   A 3- to 7-week Screening Period that includes medication washout         for subjects currently receiving testosterone treatment;     -   A 90-day randomized, open-label Treatment Period during which         subjects will receive 5.5 mg per nostril of 4.5% TBS-1 twice         daily (BID) or three times daily (TID) with potential daily dose         adjustment on Day 45 for subjects in the BID treatment group as         determined by the serum total testosterone PK profile;     -   A 90-day open-label Safety Extension Period (Safety Extension         Period 1) for all study subjects; and     -   An additional 180-day open-label Safety Extension Period (Safety         Extension Period 2) for a subset of 75 subjects.

The approximate total duration of study participation for subjects completing all 4 periods will be up to 406 days (˜58 weeks).

Screening Period

The Screening Period will take place over 3 to 7 weeks and will consist of up to 3 study visits. The duration of screening will depend on whether subjects are naïve to testosterone treatment or if they are currently being treated with a testosterone product. Subjects currently being treated with a testosterone product will require a washout. The duration of washout will depend on the type of testosterone therapy and the date of their last dose. For subjects taking testosterone injections, there must be at least 4 weeks between their last testosterone injection and the first measurement of morning serum total testosterone for qualification. For subjects taking oral, topical, or buccal testosterone, there must be at least 2 weeks between the last administration of testosterone and the first measurement of morning serum total testosterone for qualification.

Visit 1 will occur up to 7 weeks (Week −7) prior to randomization for subjects currently receiving testosterone injections, up to 5 weeks (Week −5) prior to randomization for subjects currently receiving oral, topical, or buccal testosterone, and up to 3 weeks (Week −3) prior to randomization for naïve subjects. During Visit 1, informed consent will be obtained and the subject's inclusion and exclusion criteria will be assessed based on medical interview, concomitant medications, physical examination, digital rectal examination (DRE) of the prostate, vital sign measurements, and screening laboratory evaluations. For naïve subjects, a fasting morning (0900 h±30 min) serum total testosterone level and baseline laboratory measurements will be assessed at Visit 1.

Non-naïve subjects will be instructed to discontinue all testosterone therapies at Visit 1. After Visit 1, if it is determined that a subject does not qualify for the study, the subject will be notified and instructed to restart prior testosterone therapy.

Subjects undergoing washout from testosterone therapy will return for Visit 1.1 and will have fasting morning (0900 h±30 min) serum total testosterone levels and baseline laboratory measurements obtained. For subjects undergoing washout of testosterone injections, Visit 1.1 will occur 4 weeks after the last testosterone injection (up to Week −3). For subjects undergoing washout of oral, topical, or buccal testosterone, Visit 1.1 will occur 2 weeks after the last administration of testosterone (up to Week −3). Visit 1.1 is not required for naïve subjects.

At Visit 2 (up to Week −2), all subjects will have a fasting morning (0900 h±30 min) serum total testosterone level and 12-lead electrocardiogram (ECG) assessed.

At the screening visits (Visits 1, 1.1, and 2), serum total testosterone levels will be measured using a validated assay developed by Medpace Reference Laboratories. The results will be used for determination of a subject's inclusion or exclusion from the study. To be included in the study, subjects must have 2 fasting morning (0900 h±30 min) serum total testosterone levels <300 ng/dL.

Subjects who qualify for the study based on screening assessments at Visits 1, 1.1, and 2 will be scheduled for an otorhinolaryngological (ENT) examination with nasal endoscopy performed by an ENT specialist. All qualified subjects will also have dual-energy x-ray absorptiometry scans scheduled in the interval between Visit 2 and randomization (Visit 3) for the assessment of body composition and bone mineral density.

Treatment Period

The randomized, open-label Treatment Period will consist of 4 study visits: Visit 3 (Day 1), Visit 4 (Day 30), Visit 5 (Day 60), and Visit 6 (Day 90).

Visit 3 (Day 1) will take place in the evening. At Visit 3, subjects will be randomized in a 3:1 ratio to 1 of the following 2 treatment groups:

-   -   5.5 mg per nostril of 4.5% TBS-1 BID or     -   5.5 mg per nostril of 4.5% TBS-1 TID.

Baseline levels of fasting serum total testosterone, DHT, and estradiol will be measured. Study drug (TBS-1) will be administered at 2100 h and 0700 h in the BID treatment group (total daily dose of 22 mg/day) and at 2100 h, 0700 h, and 1300 h in the TID treatment group (total daily dose of 33 mg/day). The first dose of study drug will be administered at Visit 3 (Day 1) at 2100 h and training on drug administration will be provided to subjects. Subjects will be asked to maintain a daily diary documenting administration of study drug doses throughout the Treatment Period, Safety Extension Period 1, and Safety Extension Period 2.

At Visit 4 (Day 30 to Day 31), study drug will be administered at the site, beginning with the 2100 h dose of TBS-1. Subjects will be required to remain at the site for 24 hours after the 2100 h drug administration and complete post-dose PK profiles for serum total testosterone, DHT, and estradiol will be obtained. The 24-hour C_(avg) of serum total testosterone for subjects in the BID group will be estimated based on the sum of serum total testosterone levels collected at 2 sampling points during the 24-hour PK profile: the sample collected at 9.0 hours (at 1 hour before the morning 0700 h dose) and the sample collected at 10.33 hours (20 minutes after the morning 0700 h dose). The following titration criteria will be used:

-   -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is <755 ng/dL,         then the estimated 24-hour C_(avg) is <300 ng/dL and     -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is ≥755 ng/dL,         then the estimated 24-hour C_(avg) is ≥300 ng/dL.

Subjects randomized to the BID group with an estimated serum total testosterone C_(avg)<300 ng/dL, will be contacted by phone and instructed to increase the daily dose of TBS-1 to TID on Day 45. The decision to increase the subject's daily dose to TID will be made by the investigator based on the criteria specified above. This daily dose will be continued throughout the remainder of the Treatment Period and, as applicable, both Safety Extension Periods.

At Visit 6 (Day 90 to Day 91), study drug will be administered at the site, beginning with the 2100 h dose of TBS-1. Subjects will be required to remain at the site for 24 hours after the 2100 h drug administration and complete post-dose PK profiles for serum total testosterone, DHT, and estradiol will be obtained.

At Visits 3, 4, and 6, serum total testosterone, DHT, and estradiol levels will be measured using a sensitive and specific assay developed and validated by Analytisch Biochemisch Laboratorium BV. The results will be used for PK analyses.

Safety Extension Period 1

All subjects will continue into Safety Extension Period 1 and will be instructed to continue their current daily dose of TBS-1 for the 90-day Safety Extension Period (Day 90 to Day 180). Subjects will return to the site for monthly visits.

Safety Extension Period 2

A subset of approximately 75 subjects will continue in the study for an additional 180-day Safety Extension Period (Day 180 to Day 360). The subset of subjects who continue into Safety Extension Period 2 will consist of the first subjects to complete Safety Extension Period 1. For the duration of Safety Extension Period 2, subjects will remain on the same daily dose of TBS-1 administered on Day 90 of the Treatment Period and throughout Safety Extension Period 1. Subjects will return to the site for monthly visits.

A table of the schedule of procedures can be found below:

Schedule of Procedures

Study Phase Safety Screening Treatment Period Extension Safety Extension Prior Testosterone Rando- Period 1 Period 2^(d) Testosterone Treatment miza- Efficacy Safety Subset Treatment Naive tion Analysis Analysis Safety Analysis Study Timing Day 120 Day 210 Day 300 Week-7 Day 30- Day 90- and and and or -5a Week-3 Week-2 Week-3 Week-2 Day 1 Day 31^(p) Day 60 Day 91^(q) Day 150 Day 180 Day 240 Day 270 Day 330 Day 360 Visit Number Early Study Procedures 1 1.1^(b) 2 1 2 3 4^(c) 5 6 7-8^(e) 9 10-11^(e) 12 13-14 15 Termination Inclusion/exclusion criteria x x x x x Informed consent x x Medical interview x x Physical examination x x x x x x x Height and weight x x Vital signs (HR, BP, RR, and temperature) x x x^(r) x^(r) x x^(r) x x x x x x x Concomitant medications x x x x x x x x x x x x x x x x DRE of the prostate x x x x x x x Chemistry profile and hematology^(f) x x x x x x x x x Fasting lipid profile^(g) x x x x x x x x x Liver function tests^(h) x x x x x x x x x HbA_(1c) and endocrine profile^(i) x x x x Urinalysis^(j) x x x x x x x x Urine drug and alcohol screen x x PSA x x x x x x x Estradiol and DHT^(k) x x x x x x x x Free testosterone x x x x 12-lead electrocardiogram x x x x x x x Fasting serum total testosterone^(k) x x x x x x x x x ENT exam with nasal endoscopy^(l) x x DEXA^(m) x x x x IIEF and PANAS questionnaires x x x x x^(o) Administer study drug at the site x x x 24-h PK profile for serum total testosterone, x x DHT, and estradiol Basic ENT examination (non-endoscopic) x x x x x x x x x x x Potential study drug daily dose titration x Distribute and/or review daily diary^(n) x x x x x x^(s) x x x x Weigh study drug dispensers x x x x x x x x x x x Prime study drug dispensers and distribute to x x x x x x^(s) x x x x subjects Assess adverse events x x x x x x x x x x x x x x ^(a)Visit 1 for subjects receiving intramuscular testosterone injections at the time of screening will occur at up to Week-7. Visit 1 for subjects receiving buccal, oral, or topical testosterone will occur at up to Week-5. ^(b)Visit 1.1 is only required for subjects who have undergone washout of testosterone therapy and will take place 4 weeks after the last administration of testosterone for subjects taking testosterone injections and 2 weeks after the last testosterone administration for subjects taking buccal, oral, or topical testosterone. ^(c)Based on the PK profile for serum total testosterone performed at Visit 4, some subjects in the BID treatment group will have their daily dose increased to TID. Subjects that require a daily dose increase will be contacted by phone and instructed to increase their daily dose on Day 45. ^(d)A subset of approximately 75 subjects will be enrolled in Safety Extension Period 2. ^(e)During Safety Extension Period 1 and Safety Extension Period 2, study visits will be conducted once per month. ^(f)Chemisty profile includes: creatine kinase, sodium, potassium, glucose, blood urea nitrogen, creatinine, calcium, phosphorus, and uric acid. Hematology includes: hemoglobin, hematocrit, red blood cell count, white blood cell count and differential, platelets, reticulocyte count, mean corpuscular volume, mean corpuscular hemoglobin, , and mean corpuscular hemoglobin concentration. ^(g)Fasting lipid profile includes: total cholesterol, low-density lipoprotein-cholesterol (direct), high-density lipoprotein cholesterol, and triglycerides. ^(h)Liver function tests include: total bilirubin, albumin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma glutamyl transferase. ^(i)Endocrine profile includes: thyroid-stimulating hormone, morning cortisol, sex hormone-binding globulin, luteinizing hormone, follicle-stimulating hormone, and prolactin. ^(j)Urinalysis includes: specific gravity, glucose, protein, ketones, pH, blood, bilirubin, urobilinogen, nitrite, and leukocyte esterase. ^(k)Fasting serum total testosterone, DHT, and estradiol should be collected at 0900 h ± 30 min at Visits 1, 1.1, 2, 9, 12, 15, and Early Termination and at 2045 h at Visit 3. In subjects with a known history of male hypogonadism, if 1 of the 2 serum total testosterone levels collected at screening is ≥300 ng/dL, the serum total testosterone level may be retested once. After retesting, if 2 of the 3 levels are <300 ng/dL, then the subject will be eligible to participate in the study. ^(l)ENT examination with nasal endoscopy performed by an ENT specialist will be scheduled for the interval between Visit 2 and Visit 3 (Day 1 [randomization]) on qualified subjects. ^(m)DEXA scans to evaluate body composition (total body mass, lean body mass, fat mass, and percent fat) and bone density (lumbar spine and hip) will be performed in the interval between Visit 2 and Visit 3 on qualified subjects. Follow-up DEXA will be obtained at Visit 9 (Day 180) and Visit 15 (Day 360), if scheduling is available, or within ±2 weeks of Visit 9 and Visit 15. ^(n)Daily diary will be distributed to subjects to record date and time of study drug administration. ^(o)IIEF and PANAS questionnaires will be administered to subjects at Early Termination if subjects terminate on or before Visit 6 (Day 90). ^(p)On Day 31 of Visit 4, the following procedures will be performed: vital sign measurements, basic ENT examination, administer questionnaires (may be performed on Day 30 or Day 31), and dispense daily diary. ^(q)On Day 91 of Visit 6, the following procedures will be performed: vital sign measurements, basic ENT examination, dispense daily diary, administer questionnaires (may be performed on Day 90 or Day 91), perform DRE (may be performed on Day 90 or Day 91), and perform physical examination (may be performed on Day 90 or Day 91). ^(r)At Visit 3 (Day 1), vital sign measurements will be obtained prior to first dose of study drug and at approximately 1 hour after the first dose of study drug (at 2200 h). On Day 30 of Visit 4 and Day 90 of Visit 6, vital sign measurements will be obtained once prior to administration of study drug. On Day 31 of Visit 4 and Day 91 of Visit 6, vital sign measurements will be obtained at the following approximate times after administration of study drug: 6 hours (at 0300 h), 12 hours (at 0900 h), 18 hours (at 1500 h), and 24 hours (at 2100 h). ^(s)At Visit 9, study drug dispensers and daily diaries will only be distributed to subjects entering Safety Extension Period 2. BID = twice daily; BP = blood pressure; DEXA = dual-energy x-ray absorptiometry; DRE = digital rectal examination; DHT = dihydrotestosterone; ENT = otorhinolaryngological; HbA_(1c) = glycosylated hemoglobin; HR = heart rate; IIEF = International Index of Erectile Function; PANAS = Positive and Negative Affect Schedule; PK = pharmacokinetic; PSA = prostate specific antigen; RR = respiratory rate; TID = three times daily.

Efficacy Variables Primary Efficacy Variable

The primary efficacy variable is the number and percentage of subjects with a serum total testosterone C_(avg) value within the normal range (≥300 ng/dL and ≤1050 ng/dL) on Day 90.

Secondary Efficacy Variables

Secondary efficacy variables include the following:

-   -   The number and percentage of subjects with a serum total         testosterone maximum concentration (C_(max)) value in the         following ranges on Day 90:         -   ≤1500 ng/dL,         -   ≥1800 and ≤2500 ng/dL, and         -   >2500 ng/dL;     -   The number and percentage of subjects with a serum total         testosterone C_(avg) value in the normal range (≥300 ng/dL and         ≤1050 ng/dL) on Day 30;     -   The number and percentage of subjects with a serum total         testosterone C_(max) value in the following ranges on Day 30:         -   ≤1500 ng/dL,         -   ≥1800 and ≤2500 ng/dL, and         -   >2500 ng/dL;     -   The complete PK profile (including C_(avg), the minimum         concentration, C_(max), and time to maximum concentration) of         serum total testosterone on Day 30 and Day 90;     -   The time within the normal range for serum total testosterone         based on the PK profile on Day 30 and Day 90;     -   The PK profile of serum estradiol on Day 30 and Day 90;     -   The PK profile of serum DHT on Day 30 and Day 90;     -   The ratio of DHT C_(avg) to total testosterone C_(avg) on Day 30         and Day 90;     -   The Positive and Negative Affect Schedule scores at baseline,         Day 30, Day 60, and Day 90;     -   The International Index of Erectile Function scores at baseline,         Day 30, Day 60, and Day 90;     -   Change in bone mineral density from baseline to Day 180 and from         baseline to Day 360; and     -   Change in body composition (total body mass, lean body mass, fat         mass, and percent fat) from baseline to Day 180 and from         baseline to Day 360.

Safety Variables

Safety assessments will include adverse events, clinical laboratory measurements (chemistry profile, liver function tests, fasting lipid profile, hematology, urinalysis, glycosylated hemoglobin, prostate specific antigen, and endocrine profile), 12-lead ECG parameters, vital signs (blood pressure, heart rate, temperature, and respiratory rate), physical examination parameters, DREs of the prostate, and ENT examinations.

Statistical Methodology Baseline, Endpoint, and Other Statistical Considerations

Results will be summarized by the following treatment groups:

-   -   TBS-1 BID,     -   TBS-1 BID/TID (for subjects who up-titrated at Day 45), and     -   TBS-1 TID.

For time points prior to Day 45, TBS-1 BID, TBS-1 BID/TID, and TBS-1 TID treatment groups will be presented even though no titration has occurred. Additionally, a Total TBS-1 BID treatment group (combining the TBS-1 BID and TBS-1 BID/TID groups) will be presented.

Baseline for results from the IIEF and PANAS questionnaires, vital signs, estradiol, DHT, and fasting serum total cholesterol will be the Day 1 value.

Baseline for body composition, bone mineral density, and 12-lead electrocardiogram will be the Week −2 value.

Baseline for safety laboratory results will be the Week −3 value.

If the baseline value is missing, the last value prior to the first dose of study medication will be used as baseline.

Day 90 LOCF will be the Day 90 value. If missing, the last value during the Treatment Period will be used.

Day 180 LOCF will be the Day 180 value. If missing, the last value during Safety Extension Period 1 will be used.

Day 360 LOCF will be the Day 360 value. If missing, the last value during Safety Extension Period 2 will be used.

Descriptive statistics (n, mean, standard deviation, minimum, median, maximum) will be used to summarize the continuous efficacy and safety variables. For lipids and other measurements that might violate the normal assumption, non-parametric statistics (Q1, Q3, and inter-quartile range) will be provided in addition to the conventional parametric statistics. The count and frequency will be used to tabulate the categorical measurements.

Analysis Populations Randomized Population

The randomized population will consist of all subjects who signed the informed consent form and are assigned a randomization number at Visit 3 (Day 1).

Intent-to-Treat Populations

The intent-to-treat (ITT) population for each period will consist of all subjects who receive randomized study drug and have at least one valid post-baseline efficacy measurement in the period.

Per-Protocol Population

The per-protocol population will consist of all ITT subjects who complete the 90-day Treatment Period without any major protocol deviations.

Subjects may be excluded from the per-protocol population for the following reasons:

-   -   Major violations of eligibility criteria for randomization,     -   Withdrawal Prior to Day 90 or missing Day 90 PK profile,     -   Restricted concomitant medications taken during the treatment         period, or     -   Any other major protocol deviation that may interfere with the         assessment of drug efficacy.

Safety Populations

The safety population for each period will consist of all subjects who receive randomized study drug and have safety measurements in the respective period.

Patient Disposition

Patient disposition will be summarized by counts and percentages for each treatment group and in total. The following categories of patient disposition will be included:

-   -   Subjects who are randomized,     -   Subjects who complete the Treatment Period,     -   Subjects who complete the Treatment Period and Safety Extension         Period 1,     -   Subjects who enter Safety Extension Period 2, and     -   Subjects who complete Safety Extension Period 2.

For randomized subjects who discontinue from the study, the primary reason for discontinuation will be summarized according to the period in which the withdrawal occurred. Reasons for discontinuation will be listed.

The total number of subjects who are screened and the total number of screen failures with reasons for screen failure will be tabulated.

The number and percentage of subjects in the ITT populations, PP population, and safety populations will be presented by treatment group and in total.

Demographic and Baseline Characteristics

Demographic and baseline characteristics will be summarized for all subjects in the randomized population by treatment group and in total.

Gender, race, testosterone therapy history, smoking status, and alcohol use will be summarized with counts and percentages. Age, height, weight, body mass index (BMI), and duration of hypogonadism will be summarized with descriptive statistics.

Baseline values for fasting serum total testosterone will be described with descriptive statistics.

Baseline is defined in Section 0

Baseline, Endpoint, and Other Statistical Considerations.

Medical history will be listed for all randomized subjects.

Prior/Concomitant Medications

Medication start and stop dates that are recorded on the Prior and Concomitant Medications Case Report Form (CRF) will be used to determine whether the medications are prior or concomitant to the treatment and safety extension periods. Prior medications are defined as those used prior to and stopped before the first dose of study medication.

Concomitant medications are those that are used during the treatment period or safety extension periods (i.e., start date is on or after the first dose date of study medication, or start prior to the date of first dose and the stop date is either after the first dose date or marked as “continuing”).

Concomitant medication/therapy verbatim terms will be coded with Anatomical Therapeutic Chemical (ATC) class and preferred term by the World Health Organization

Drug Dictionary. The numbers and percentages of subjects in each treatment group taking concomitant medications will be summarized by ATC class and preferred term for the safety population for the Treatment Period. Concomitant medications taken during Safety Extension Period 1 and Safety Extension Period 2 will be summarized in a similar manner.

Prior and concomitant medications will be listed.

Study Exposure, Dispensation, and Accountability

Days of exposure to study medication during the Treatment Period, Safety Extension Period 1, and Safety Extension Period 2 will be summarized with descriptive statistics for the safety populations for each treatment group and overall. Contingency tables will be provided to display the number and percentage of subjects with exposure by visit for each treatment group for the safety populations.

Days of exposure is defined as the date of the last dose of study medication (in the respective period)−the date of the first dose of study medication+1.

Drug dispensation and accountability data will be listed.

Analysis of Efficacy

Efficacy evaluations will be performed for the ITT populations. The primary efficacy analysis will be repeated for the PP population.

Analysis of the Primary Efficacy Parameter

The primary objective of this study is to determine the efficacy of 4.5% TBS-1 gel, administered intranasally BID and/or TID, in increasing the C_(avg) of serum total testosterone to the normal range (≥300 ng/dL and ≤1050 ng/dL) in male subjects with hypogonadism after 90 days of treatment. The primary efficacy parameter, C_(avg), will be calculated from the AUC using the following formula:

C _(avg)=AUC_(0-24h)/24.

The AUC curve for both the BID and TID dosing regimens will be determined for the 0-24 hour time interval by using linear trapezoidal and linear interpolation methods. Actual collection times will be used in the calculation.

The number and percentage of subjects who reach the treatment goal (ie, serum total testosterone C_(avg) value in the normal range) at Day 90 or Early Termination (Day 90 LOCF) will be summarized by treatment group. 95% confidence intervals for the frequency will be approximated by a binomial distribution within each treatment group.

Analysis of the Secondary Efficacy Parameters

The primary efficacy analysis will be repeated for the serum total testosterone C_(avg) values on Day 30. Additionally, for C_(avg) on Day 30, the Total BID treatment group and the TID treatment group will be compared using the chi-square test to evaluate the number of subjects with C_(avg) within the normal range (≥300 ng/dL and ≤1050 ng/dL).

The odds ratio, 95% confidence interval, and p-value will be presented.

The serum total testosterone C_(max) values on Day 30 and Day 90 will be summarized by counts and percentages for each treatment group for the following categories:

-   -   C_(max)≤1500 ng/dL,     -   1800 ng/dL≤C_(max)≤2500 ng/dL, and     -   C_(max)>2500 ng/dL.

The PK profile, including AUC_(0-24h), C_(avg), C_(min), C_(max), and T_(max), for serum total testosterone, serum estradiol, and serum DHT will be summarized with descriptive statistics, including the arithmetic mean, standard deviation, coefficient of variation (CV %), geometric mean, median, minimum, and maximum by treatment at Day 30 and Day 90. Geometric mean and CV % will not be presented for T. The same descriptive statistics will be calculated for serum concentrations at each sampling time by treatment and visit.

Data will be listed individually for all subjects. A figure displaying the distribution of the C_(avg) values at Day 30 and Day 90 will be provided.

All concentrations below the lower limit of quantification (LLOQ) or missing data will be labeled as such in the concentration data listings. Concentrations below the LLOQ prior to the first measurable concentration will be treated as zero in the summary statistics and for the calculation of PK profile parameters. Concentrations below LLOQ after the time point of the first measurable concentration will be set to missing and not included in the calculation of AUC.

The time within normal range (≥300 ng/dL and ≤1050 ng/dL) for serum total testosterone and the ratio of DHT C_(avg) to total testosterone C_(avg) on Day 30 and Day 90 will be summarized with descriptive statistics for each treatment group.

The concentrations of fasting serum total testosterone, DHT, and estradiol will be summarized with descriptive statistics at baseline, Day 30, Day 90, Day 90 LOCF, Day 180, Day 180 LOCF, Day 270, Day 360, and Day 360 LOCF. The change from baseline will also be summarized.

The change in bone mineral density, total body mass, lean body mass, fat mass, and percent fat will be summarized with descriptive statistics at baseline, Day 180, and Day 360, as well as the change from baseline to Day 180 and the change from baseline to Day 360 for each treatment group.

The Day 30 24-hour C_(avg) serum total testosterone values for all subjects in the BID treatment group will be assessed for appropriate dose titration (from BID to TID) at Day 45.

The IIEF questionnaire will be broken up into five domains: erectile function, intercourse satisfaction, orgasmic function, sexual desire, and overall satisfaction. Point values will be assigned to each answer in the questionnaire according to Appendix 1. (See Appendix 1 in the Appendix to the Specification.) Domain scores will be the sum of the points of each question making up the domain. The breakdown can be found in the table below.

Domain Questions Maximum Score Erectile Function 1, 2, 3, 4, 5, 15 30 Intercourse 6, 7, 8 15 Satisfaction Orgasmic Function 9, 10 10 Sexual Desire 11, 12 10 Overall Satisfaction 13, 14 10

The scores for each domain will be summarized with descriptive statistics at baseline, Day 30, Day 60, Day 90, Day 90 LOCF, and the change from baseline at each visit.

PANAS scores will be summarized with descriptive statistics for each emotion/feeling as well as the Positive and Negative Affect Score by treatment at baseline, Day 30, Day 60, Day 90, and Day 90 LOCF. Change from baseline to each visit will be provided for the Positive and Negative Affect Scores. Positive Affect Score is found by adding the scores from items 1, 3, 5, 9, 10, 12, 14, 16, 17, and 19. Negative Affect Score is found by adding the scores from items 2, 4, 6, 7, 8, 11, 13, 15, 18, and 20. A separate summary will be performed to summarized the PANAS scores based on how the subject ‘felt over the past week’, not including those scores based on how the subject ‘feels right now’.

Analysis of Safety

All analyses of safety will be conducted on the safety populations and will be summarized by treatment group and in total. The safety assessments include adverse events, clinical laboratory measurements, DRE of the prostate, 12-lead ECGs, vital sign measurements, basic ENT examination, and physical examination.

Adverse Events

An adverse event (AE) is defined as any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related. An adverse event can therefore be any unfavorable and/or unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of an investigational medication product, whether or not related to the investigational medication product. All adverse events, including observed or volunteered problems, complaints, or symptoms, are to be recorded on the appropriate eCRF. AEs will be coded using the latest version of MedDRA.

Treatment-emergent adverse events (TEAEs) are defined as those AEs that have a start date on or after the first dose of randomized study medication, or occur prior to the first dose and worsen in severity during the treatment period. Drug-related AEs are defined as those AEs with relationship to study drug as “Probable” or “Definitely Related”.

TEAEs will be summarized in which period the AE began. For example, TEAEs during Safety Extension Period 1 will be any TEAEs that occur on or after the first day of Safety Extension Period 1 through the end of the study or the start of Extension Period 2.

A table overview of adverse events will be provided summarizing the counts and percentages of subjects with the following adverse events during the Treatment Period:

-   -   TEAEs,     -   Maximum severity of TEAEs,     -   Drug-related TEAEs,     -   Maximum severity of drug-related TEAEs,     -   All serious adverse events (SAEs),     -   All treatment-emergent SAEs,     -   Drug-related SAEs,     -   Death due to AEs,     -   Withdrawals due to AEs, and     -   Withdrawals due to drug-related AEs.

A similar overview for TEAEs with onset date during Safety Extension Period 1 and Safety Extension Period 2 will be provided.

The counts and percentages of subjects with TEAEs during the Treatment Period will be summarized for each treatment group by system organ class and preferred term. Drug-related TEAEs, SAEs, and TEAEs leading to discontinuation of study medication during the Treatment Period will be summarized in the same manner. Summaries of maximum severity for TEAEs and drug-related TEAEs will be provided.

The counts and percentages of subjects with TEAEs during Safety Extension Period 1 and Safety Extension Period 2 will be summarized for each treatment group by system organ class and preferred term. Drug-related TEAEs will be summarized in the same manner.

All SAEs and TEAEs leading to discontinuation of study medication will be listed with detailed information.

Clinical Laboratory Assessments

Continuous laboratory results for selected laboratory parameters (including hematology, chemistry, urinalysis, lipid profile, liver function tests, HbA_(1c) and endocrine profile) will be presented by treatment group and summarized with descriptive statistics for each scheduled visit and for the end of each period. The change from baseline will also be presented.

Categorical laboratory results will be presented with the frequency and percentage in each category by treatment group for each scheduled visit and for the end of each period.

The number and percentage of subjects with laboratory abnormalities will be summarized by treatment group and overall for each period. The worst value for each subject in each period will be summarized.

Listings will be provided for all laboratory parameters.

Physical Examination, Digital Rectal Exam, ENT Exam, and Nasal Endoscopy

Physical examination findings will be summarized by treatment group with counts and percentages for each body system for each scheduled visit and for the end of each period. Digital rectal exam, ENT examination, and nasal endoscopy results will be summarized in a similar manner.

Physical examination, digital rectal exam, ENT exam, and nasal endoscopy findings will be listed by subject.

Weight, BMI, Vital Signs, and 12-Lead Electrocardiogram

Weight, BMI, vital signs, and quantitative ECG parameters (Heart Rate, PR Interval, QRS Interval, and QT Interval) will be summarized with descriptive statistics at baseline, each post-baseline visit, and the end of each period. The change from baseline will also be presented. Counts and percentages of subjects with abnormal ECG results will be tabulated.

Vital signs recorded during the PK sampling and overall interpretations from ECG will be listed.

Report Analyses

Two report analyses will be generated for this study.

The first analysis will be conducted after all subjects complete the Treatment Period.

The analysis will include all primary and secondary efficacy endpoints. Safety data collected through Safety Extension Period 1 will also be summarized.

After all subjects complete the study, including Safety Extension Period 2, a second analysis will be generated including all safety and efficacy data.

Sample Size Determination

A sample size of approximately 280 subjects (210 subjects randomized to the BID treatment group and 70 subjects randomized to the TID treatment group) was selected to provide a sufficient number of subjects to determine the efficacy, safety, and tolerability of 4.5% TBS-1 gel. Since this is an observational study, no formal sample size calculation was performed.

Programming Specifications

The programming specifications, including the mock-up validity listings, analysis tables, figures, and data listings, as well as the derived database specifications, will be prepared in stand-alone documents. The programming specification documents will be finalized prior to database lock.

LIST OF ABBREVIATIONS AND DEFINITION OF TERMS

ALT Alanine transaminase

AST Aspartate transaminase

AUC Area under the curve

BID Twice daily

C_(avg) Average concentration

C_(max) Maximum concentration

C_(min) Minimum concentration

CRA Clinical research associate

CTIVRS ClinTrak™ Interactive Voice Response System

DEXA Dual-energy x-ray absorptiometry

DHEA Dehydroepiandrosterone

DHT Dihydrotestosterone

DRE Digital rectal examination

ECG Electrocardiogram

eCRF Electronic case report form

EDC Electronic data capture

ENT Otorhinolaryngological

FSH Follicle-stimulating hormone

GnRH Gonadotropin-releasing hormone

H2 Histamine 2

HbA_(1c) Glycosylated hemoglobin

IIEF International Index of Erectile Function

IRB Institutional Review Board

ITT Intent-to-treat

LH Luteinizing hormone

MedDRA Medical Dictionary for Regulatory Activities

PANAS Positive and Negative Affect Schedule

PDE5 Phosphodiesterase 5

PK Pharmacokinetic

PSA Prostate specific antigen

SAE Serious adverse event

SHBG Sex hormone-binding globulin

TID Three times daily

TWNR Time within the normal range

t_(1/2) Half-life

T_(max) Time to maximum concentration

TSH Thyroid-stimulating hormone

TU Testosterone undecanoate

ULN Upper limit of normal

See Appendix 1 in the Appendix to the Specification Example 15 Titration Method for Dosing BID or TID Intranasal Testosterone Gels

The present invention is also concerned with a novel titration method to determine the appropriate daily treatment regimen, i.e., a BID or TID treatment regimen, to administer the intranasal gels of the present invention to treat hypogonadism or TRT. While the preferred treatment regimen in accordance with the present invention for administering the intranasal testosterone gels, such as 4.0% or 4.5% TBS-1 as described in Examples 1, 2, 3, 5, 7, 8, 9 and 10 above, to treat hypogonadism or TRT is twice-daily (BID) treatment regimen, the present invention contemplates that certain subjects may be more effectively treated with a three-times-a-day (TID) treatment regimen. Thus, the novel titration method of the present invention has been developed to determine which subject will require a BID or TID treatment regimen to more effectively treat hypogonadism or TRT when treated with the intranasal testosterone gels of the present invention. See also Exhibit C (the contents of which are incorporated herein by reference).

In carrying out the novel titration method in accordance with the present invention, subjects will have 2 blood draws, preferably at 7 am and at 8:20 am on the test day. The day before the first blood draw, the subject will take at 10 pm, his evening intranasal dose of TBS-1. On test day, the subject will take at about 8 am, his morning intranasal dose of TBS-1.

The 24-hour C_(avg) of serum total testosterone will be estimated based on the sum of serum total testosterone levels collected at the 2 sampling points: the sample collected at about 9.0 hours (at 7 am, which is 1 hour before the morning 0800 h intranasal dose) and the sample collected at about 10.33 hours following the last evening's intranasal dose (20 minutes after the morning 0800 h dose+/−20 minutes). Note that, the blood draw times may be changed (+/−1 hour) but the delay between the last dose and the first blood draw is preferably 9 hours+/−20 minutes and the delay between the next dose administered at about 10 hours+/−20 minutes after the last dose and the second blood draw is preferably +/−20 minutes.

Testosterone serum concentrations are preferably measured by a validated method at a clinical laboratory and reported in ng/dL units.

The following titration criteria is preferably used:

-   -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is <755 ng/dL,         then the estimated 24-hour C_(avg) for the male patient is <300         ng/dL     -   If the sum of the serum total testosterone level values for PK         samples collected at 9.0 hours and 10.33 hours is ≥755 ng/dL,         then the estimated 24-hour C_(avg) for the male patient is ≥300         ng/dL.

With respect to those subjects with an estimated serum total testosterone C_(avg)<300 ng/dL, i.e., those subjects who sum of the serum total testosterone level values for PK samples collected at 9.0 hours and 10.33 hours is <755 ng/dL, their BID treatment regimen should be titrated to a TID treatment regimen of TBS-1 to achieve a 24-hour C_(avg) of ≥300 ng/dL. The decision to titrate the subject's daily dose to TID, however, will be made by the doctor based on the criteria specified above.

With respect to those subjects with an estimated serum total testosterone C_(avg)≥300 ng/dL, i.e., those subjects who sum of the serum total testosterone level values for pK samples collected at 9.0 hours and 10.33 hours is ≥755 ng/dL, their BID treatment regimen should remain unchanged at a BID treatment regimen of TBS-1 since their 24-hour C_(avg) is ≥300 ng/dL. The decision to titrate the subject's daily dose to TID or remain at BID, however, will be made by the doctor based on the criteria specified above.

It should be understood that, while it is preferred to draw blood from a subject to test the subject's serum total testosterone level values for pK samples at 9 hours and at 10.33 hours after the last evening's BID dose, the difference in the total draw time, i.e., 10.33 hours, may vary by as much as about +/−60 minutes and preferably no more than about +/−20 minutes between one another. It should also be understood that while, serum total testosterone level values for PK samples is 755 ng/dL is the preferred level to use to determine if titration to TID is necessary, the serum total testosterone level values for PK samples may vary as much as +/−50 and preferably no more than +/−25.

As an alternative, it should be understood that, while the titration method is described above with starting the titration method based upon the last evening's BID dose, the titration method could also be used by starting the titration method based upon the first morning dose. For example, under this alternative embodiment, the first blood draw would be taken at about 9 hours and the second blood draw would be taken at about 10.33 hours after the morning dose, so long as the second blood draw is taken at about 20 minutes after the last BID dose of the day.

Phase III Study—Rationale for the Titration Protocol for Compleo (4.5% TBS-1 Gel) 1. Introduction

At the Mar. 14, 2011 End of Phase II Meeting, the Compleo (4.5% TBS-1 Gel) Phase

III study includes the modifications suggested by the Agency (“FDA”) and a rationale for the choice of secondary endpoints, the titration scheme and the ENT examination protocol. See Example ______ for the final Phase 3 protocol.

The primary endpoint of this study is the percentage of subjects with a serum total testosterone C_(avg) value within the normal range on Day 90. This endpoint is consistent with Agency standards used for approval of other testosterone replacement therapy formulations. Although there are no generally accepted lower limits of normal for serum total testosterone, guidelines recommend using the range of 280-300 ng/dL. The sponsor has defined the normal range for Testosterone as 300 ng/dL to 1050 ng/dL for this study. This range is consistent with Agency standards and is in agreement with the AACF Hypogonadism and Endocrine Society Clinical Practice Guidelines.

Secondary Endpoints

The secondary endpoints in the Compleo (4.5% TBS-1 Gel) Phase III study and the rationale are listed below and included in the final protocol. All of the secondary endpoints proposed are well established for testosterone replacement therapies.

DHT—

In previous trials with Compleo, following the administration of Compleo, the DHT levels of responders were increased from below normal to within the normal range. These levels remained stable within the normal range during the treatment and returned to basal levels after discontinuation of Compleo. The upper limit of the physiological reference range of DHT was not achieved or exceeded by any subjects for any treatment. As DHT is the major metabolite of Testosterone, an increase in DHT to within the normal range is evidence of Testosterone replacement. A full DHT pharmacokinetic profile will be collected at Day 30 and 90 for comparison against the baseline levels.

Body Composition and Lean Body Mass—

The effect of testosterone replacement therapy on body composition and lean body mass has been included as an additional objective measurement of efficacy. The sponsor will use DEXA to evaluate the subjects for this criteria at baseline and Day 90.

Bone Mineral Density—

This parameter will be measured by DEXA at baseline and Day

Erectile Function—

Erectile function was included in the proposed protocol but based on the recommendations from the Division, erectile function will now be assessed using the IIEF (International Index of Erectile Function Questionnaire),

Mood Scales—

The sponsor intends to collect data on changes in subject mood compared to baseline using the PANAS scale for information purposes only. The PANAS scale was chosen as it is a validated instrument that measures the balance between positive and negative mood. Data will be collected for each subject at baseline, Day 30 and Day 90.

Study Design

The study includes a fixed dose arm for the t.i.d. administration and the previously proposed b.i.d. titration arm. The subjects in the b.i.d. group will be evaluated at Day 30 in accordance with the established titration scheme and those subjects that require titration will be titrated to t.i.d. dosing. The subjects in the b.i.d. group that are not titrated will constitute a second fixed dose arm for b.i.d. dosing.

The sample size has been modified accordingly to ensure that sufficient subjects are available for the safety evaluation. The new sample size of 280 subjects will be split into two groups, with 210 subjects randomized to the b.i.d. titration treatment group and 70 subjects randomized to the t.i.d. treatment group. The sample size (see Table 1) incorporates a 50% titration rate from b.i.d. to t.i.d., a 75% responder rate for all t.i.d. patients and a 20% drop out rate.

TABLE 1 Sample Size Estimation Randomization Arm b.i.d. b.i.d/t.i.d. t.i.d. Number of Subjects 210 — 70 Number Post- 105 105 70 titration (50% titration rate) Responder Rate at 100% 75% 75% Day 90 Drop Outs  20% 20% 20% Total Subjects for 84 63 42 Safety Evaluation

Titration Scheme Titration Model Development

Following the discussion with the Division the recommendation to prospectively develop a titration scheme and include this in the Phase III study has been adopted. The titration scheme, based on two individual blood levels, has been designed to consistently titrate subjects from the b.i.d. treatment group to the t.i.d. treatment group, when testosterone replacement is not being achieved with b.i.d. dosing. Two hundred and ten (210) subjects will be randomized to the b.i.d. treatment group. Subjects will receive Compleo at 2100 h and 0700 h. On Day 30, all subjects will be required to remain at the site for 24 hours after drug administration to obtain a 24 hour pharmacokinetic profile, actual C_(avg). Although a 24 hour profile will be taken, the full profile will not be used for titration decisions. A titration scheme has been developed to allow for a simple and consistent assessment of each subject.

A number of different models were examined in the development of the titration scheme for Compleo that included both single and multiple analysis points. The model fit development and subsequent analysis was completed based on the data from the TBS-1-2010-01 study.

The model selected uses two testosterone measurements, one taken one hour prior to the morning dose (sample A, 9.00 h post 1^(st) dose) and one taken 20 minutes after the morning dose (sample B, 10.33 h (10 h20 min) post 1^(st) dose). The C_(avg) for a given subject was predicted using a ratio of the two testosterone measurements triangulated to predict the area under the curve for the morning peak. This morning peak area was used to predict the total area under the curve for a 24 hour dosing interval which was converted to the 24 hour C_(avg) for testosterone. This is referred to as the ‘model predicted C_(avg)’ or ‘calculated C_(avg)’ in this text. The calculated C_(avg) was then compared against the lower limit of normal of 300 ng/dL as the decision level for titration. If the C_(avg) is calculated to be greater than 300 ng/dL then the b.i.d. regimen is maintained. If the C_(avg) is calculated to be less than 300 ng/dL then the patient is titrated to the t.i.d. regimen. The individual data comparing the predicted C_(avg) with the actual C_(avg) C_(avg) is provided in Appendix 2.

The model was further challenged on simulated pharmacokinetic profile data from 200 patients based on the 11 mg b.i.d. treatment group from the TBS-1-2010-01 study. Using the sampling points from the model and the individual subject profiles from these 200 subjects, a model predicted C_(avg) was calculated and compared to the actual C_(avg). The individual data from this analysis is provided in Appendix 3. The model was designed to have a high degree of precision, (successful prediction rate of greater than 80%) around the decision level of 300 ng/dL, and the data from both datasets shows a good correlation between the predicted C_(avg) and the actual C_(avg) around this key decision level.

The titration model was used to create a titration scheme that will be utilized and challenged in the Phase III study. This scheme uses the two sampling points from the model; one sample taken one hour prior to the morning dose (Sample A) and one sample taken 20 minutes after the morning dose (Sample B). If the sum of Sample A and Sample B is 755 ng/dL or greater, the 24 hour testosterone C_(avg) is predicted to be greater than 300 ng/dL and titration is not required. If the sum of Sample A and Sample B is less than 755 ng/dL, the 24 hour testosterone C_(avg) is predicted to be lower than 300 ng/dL and titration is required.

Titration Model Robustness

The robustness of the model and the resulting titration scheme was evaluated using the data from the TBS-1-2010-01 study and the 200 patient simulated subject profiles. In addition to the two sampling points in the model, analysis was performed on three other sampling timepoints after the morning dose, 30 minutes, 60 minutes and 90 minutes. In each case the titration scheme was used to predict the requirement for titration following which each subject was sorted into one of two groups, Titration Required or Titration Not Required. The actual C_(avg) for each subject was used to assess the accuracy of the titration scheme with the total number of correct and incorrect titration predictions determined. The incorrect predictions were further separated into two groups in which:

-   -   (1) Titration was not predicted but required—The titration         scheme indicated that titration was not required, whereas the         actual C_(avg) was less than 300 ng/dL. The subjects would not         be titrated.     -   (2) Titration was predicted but not required—The titration         scheme indicated that titration was required, whereas the actual         C_(avg) was greater than 300 ng/dL. The subjects would be         titrated.

The data for this analysis is shown in Table 2. The individual model data for each of the different sample timepoints can be found in Appendix 4, 5 and 6.

TABLE 2 Success Ratio for the Titration Model Analysis based on a Comparison of Predicted and Actual 24 hour Average Concentration Values (C_(avg)) Model Dataset and Sampling Time TBS-1-2010-1 TBS-1-2010-1 Simulated Simulated Data Data Sample A)-1 hr Sample A)-1 hr TBS-1-2010-1 TBS-1-2010-1 TBS-1-2010-01 before AM before AM Simulated Data Simulated Data Sample A)-1 hr dosing dosing Sample A)-1 hr Sample A)-1 hr before AM dosing Sample B) + 20 Sample B) + 40 before AM dosing before AM dosing Sample B) + 20 min min after AM min after AM Sample B) + 60 mm Sample B) + 90 min Success Criteria after AM dose dose dose after AM dose after AM dose Number of 22 200 200 200 200 Subjects Evaluated Total Correct (%) 18 (81.8%) 169 (84.5%) 165 (82.5%) 144 (72.5%) 147 (73.5%) Titration 3 36 37 38 43 Required 15 133 128 106 104 Not Titrated Titration predicted  4 (18.2%)  23 (11.5%)  26 (13.0%)  44 (22.0%)  50 (25.0%) but not required (%) Titration not 0 (0.0%) 8 (4%)  9 (4.5%)  7 (3.5%)  3 (1.5%) predicted but required (%)

As the data indicates the model is capable of predicting the need for titration on a consistent basis with an over 80% success ratio for correct predictions at the proposed sampling points. This holds true for the sampling point at 40 minutes after the morning dose as well. At sampling points 60 minutes and 90 minutes after the morning dose the prediction success falls below 80%, which is likely explained by the variability of the values in testosterone concentration at these timepoints and the added variability introduced by the simulation analysis. The model performs slightly better using the 90 minute sampling point than the 60 minute sampling point.

accurately identify those subjects that would benefit from titration from the b.i.d. to the t.i.d. dosing regimen and, in doing so, kept the number of subjects for which titration was not predicted but required to a minimum. The titration scheme achieved this with very low numbers of subjects from the TBS-1-2010-01 study data and TBS-1-2010-01 simulation data across all post dose timepoints.

The remaining subjects that were not correctly predicted by the titration scheme were titrated when it was not necessarily required. Based on the safety and pharmacokinetic profile data from the TBS-1-2010-01 study, none of the subjects that were on a t.i.d. regimen of 4.5% gel (33.75 mg/day) showed any supra-physiologic levels for testosterone or high Cmax values, meaning there is no safety concern with subjects who are titrated to t.i.d. when they were achieving acceptable testosterone levels on b.i.d. treatment.

Titration Scheme Validation

By including the titration scheme in the Phase III study and correlating the titration decision made with the actual measured C_(avg) on Day 30 for each subject in the b.i.d. group at the end of the study, the exercise performed above on the simulation data will be repeated to evaluate and assess the accuracy of the titration. This internal validation will serve to support the validation scheme as proposed or provide the necessary information required to make any modifications for the product label.

ENT Evaluation During Safety Assessment

The detailed synopsis has been updated to clarify the procedure and criteria for the ENT evaluation that will be included in the safety extension for the Phase III study. As previously agreed, a long-term safety assessment will be performed; 200 subjects will be exposed for an addition 3 months and 50 subjects will be exposed for an additional 6 months.

The purpose of the ENT examination is to determine if there have been any adverse reactions related to the nasal cavity that were caused by either the study drug or the multiple dose dispenser. A trained physician will perform the ENT examination as described.

1. History:

The examining physician will inquire about the following symptoms:

-   -   Excessive nasal dryness,     -   Excessive nasal crusting,     -   Unexpected nasal bleeding,     -   Progressive nasal pain,     -   Progressive nasal obstruction, and     -   Alternation to sense of smell.

2. Physical exam:

-   -   Using an anterior rhinoscope with a headlight or other light         source, the trained physician will look for the following:         -   Large amounts of nasal crusting,         -   Scar tissue blocking the nose,         -   Dried or fresh nasal blood, and         -   Fissuring of the nasal skin.

The safety evaluation measures (Day 90 to 180) will consist of monthly ENT examinations, vital signs and adverse events assessments. On Day 180 (or early termination), subjects will also undergo a physical examination, 12-lead ECG, DRE of the prostate, and laboratory assessments (CBC, PSA, chemistry profile, liver function tests, lipid profile, urinalysis, fasting morning serum total testosterone, DHT, and estradiol).

A subset of subjects will be asked to continue in the study for a safety extension period (Day 180 to 360). In this period, safety evaluations will consist of monthly ENT examinations, vital signs, and adverse events. On Day 270 and Day 360 (or early termination), subjects will also undergo a physical examination, 12-lead ECG, DRE of the prostate, and laboratory assessments (CBC, PSA, chemistry profile, liver function tests, lipid profile, urinalysis, fasting morning serum total testosterone, DHT, and estradiol).

Titration Model Results-TBS-1-2010-01 Data Testosterone Model Actual Concentration (ng/dL) Predicted 24 h Subject Time From Morning Dose 24 h Cavg Cavg Number −1 hr +20 min (ng/dL) (ng/dL) C1 399 600 428 518 C2 251 327 248 360 C3 344 604 406 387 C4 463 648 476 429 C5 292 558 364 410 C6 161 304 199 179 C7 316 1140 624 489 A1 284 482 328 372 A2 249 363 262 308 A3 303 611 392 337 A4 216 549 328 325 AS 552 872 610 523 A6 320 671 425 603 A7 347 979 568 582 A8 185 424 261 248 B1 308 847 495 369 B2 333 1100 614 446 B3 226 492 308 311 B4 249 658 389 351 B5 195 472 286 295 B6 446 959 602 723 B7 355 370 311 359

Titration Model Results TBS-1-2010 Patient Simulation Data Testosterone Sample A taken 1 hour before the morning dose Testosterone Sample B taken 20 minutes after the morning dose Testosterone Concentration Model (ng/dL) Predicted Actual Subject Time From Morning Dose 24 h Cavg 24 h Cavg Number −1 hr +20 min (ng/dL) (ng/dL) 1 217 879 433 501 2 286 724 399 393 3 406 296 278 358 4 449 665 440 392 5 367 561 367 514 6 254 566 324 334 7 406 832 489 584 8 483 639 444 450 9 108 339 177 175 10 260 827 430 366 11 327 550 347 322 12 522 1430 772 757 13 240 995 488 442 14 278 852 447 288 15 249 448 276 330 16 523 930 574 506 17 497 726 483 423 18 375 945 522 412 19 132 278 162 291 20 231 439 265 306 21 363 844 477 505 22 535 770 516 505 23 340 680 403 673 24 252 630 349 346 25 231 583 322 322 26 156 430 232 362 27 226 719 374 375 28 197 485 270 339 29 466 635 435 459 30 232 920 455 316 31 263 897 459 431 32 183 1260 570 352 33 491 655 453 436 34 277 497 306 475 35 362 403 302 441 36 140 395 212 233 37 164 410 227 301 38 195 374 225 209 39 381 904 508 527 40 206 464 265 316 41 176 809 389 397 42 200 485 271 282 43 479 2640 1233 877 44 280 511 313 332 45 212 705 363 315 46 246 436 270 300 47 117 358 188 203 48 420 679 434 308 49 262 781 412 406 50 191 688 347 359 51 381 447 327 439 52 321 553 346 422 53 195 424 245 397 54 325 425 296 301 55 413 895 517 472 56 179 952 447 328 57 180 472 258 249 58 206 689 354 359 59 255 563 323 318 60 380 683 420 437 61 226 358 231 317 62 370 628 395 388 63 265 885 455 388 64 163 475 252 245 65 302 756 418 595 66 274 597 344 377 67 250 666 362 456 68 383 789 463 456 69 291 662 377 411 70 430 1060 589 417 71 236 514 296 307 72 248 550 315 248 73 244 692 370 374 74 362 711 424 572 75 310 1370 664 589 76 467 475 372 367 77 256 465 285 236 78 222 557 308 267 79 198 727 366 333 80 371 397 304 346 81 213 993 477 549 82 288 646 369 386 83 221 538 300 314 84 226 574 316 355 85 168 679 335 286 86 139 507 255 334 87 211 611 325 363 88 225 686 360 412 89 265 456 285 313 90 520 1140 656 510 91 207 1120 525 365 92 191 515 279 304 93 242 689 368 399 94 121 916 410 331 95 105 398 199 294 96 299 686 389 348 97 153 466 245 441 98 242 417 261 260 99 288 697 389 448 100 530 720 494 494 101 178 685 341 211 102 410 424 330 343 103 491 646 449 401 104 443 830 503 408 105 218 489 279 314 106 322 526 335 347 107 404 660 421 420 108 217 491 280 281 109 277 454 289 294 110 200 281 190 229 111 258 425 270 276 112 523 642 461 440 113 283 568 336 408 114 260 596 338 242 115 231 600 329 389 116 481 757 489 405 117 293 543 330 370 118 261 375 251 311 119 152 226 149 227 120 291 412 278 282 121 383 547 368 440 122 295 352 256 295 123 276 527 317 304 124 113 1100 480 311 125 245 421 263 307 126 421 666 430 499 127 313 514 327 371 128 238 787 405 432 129 256 493 296 380 130 175 278 179 278 131 199 394 234 303 132 296 491 311 363 133 319 879 474 342 134 274 812 429 346 135 140 412 218 220 136 466 441 359 434 137 449 2330 1099 739 138 252 347 237 282 139 330 698 406 440 140 165 513 268 215 141 292 667 379 389 142 306 574 348 473 143 345 319 263 292 144 359 880 490 492 145 399 561 380 449 146 593 932 603 696 147 581 712 511 426 148 222 356 229 229 149 231 640 344 301 150 274 1160 567 460 151 178 764 372 411 152 176 1030 477 312 153 346 542 351 491 154 221 378 237 259 155 236 527 302 361 156 257 456 282 358 157 146 609 298 295 158 241 358 237 304 159 199 636 330 434 160 280 1220 593 506 161 370 500 344 417 162 148 710 339 250 163 246 402 256 284 164 263 930 472 349 165 178 537 283 275 166 393 715 438 432 167 193 426 245 246 168 148 449 236 214 169 120 596 283 217 170 262 593 338 307 171 369 652 404 366 172 394 666 419 415 173 289 543 329 396 174 282 548 328 329 175 225 654 347 336 176 255 970 484 544 177 212 548 300 344 178 203 251 179 201 179 488 612 435 560 180 247 809 417 425 181 206 565 305 280 182 134 497 249 252 183 401 442 333 368 184 253 550 317 329 185 252 843 433 352 186 314 677 392 494 187 370 532 357 378 188 368 551 363 342 189 241 511 297 251 190 229 794 404 379 191 333 636 383 526 192 217 834 415 360 193 264 411 267 263 194 382 1180 618 479 195 283 380 262 344 196 332 1360 669 442 197 310 911 483 321 198 194 610 318 327 199 172 465 252 192 200 335 894 486 473

See FIG. 40,

Titration Model Results TBS-1-2010 Patient Simulation Data Testosterone Sample A taken 1 hour before the morning dose Testosterone Sample B taken 40 minutes after the morning dose Testosterone Model Concentration (ng/dL) Predicted Actual Subject Time From Morning Dose 24 h Cavg 24 h Cavg Number −1 hr +40 min (ng/dL) (ng/dL) 1 217 1070 509 501 2 286 962 493 393 3 406 399 318 358 4 449 676 445 392 5 367 1420 706 514 6 254 863 441 334 7 406 991 552 584 8 483 758 490 450 9 108 229 133 175 10 260 441 277 366 11 327 432 300 322 12 522 1160 665 757 13 240 565 318 442 14 278 417 275 288 15 249 474 286 330 16 523 1020 610 506 17 497 992 588 423 18 375 1130 595 412 19 132 379 202 291 20 231 439 256 306 21 363 858 483 505 22 535 770 476 505 23 340 630 383 673 24 252 630 366 346 25 231 1130 538 322 26 156 736 353 362 27 226 456 270 375 28 197 324 206 339 29 466 467 369 459 30 232 348 229 316 31 263 1170 566 431 32 183 603 311 352 33 491 660 455 436 34 277 426 278 475 35 362 334 275 441 36 140 485 247 233 37 164 568 289 301 38 195 374 225 209 39 381 1020 554 527 40 206 410 243 316 41 176 588 302 397 42 200 651 336 282 43 479 1750 881 877 44 280 686 382 332 45 212 524 291 315 46 246 353 237 300 47 117 532 256 203 48 420 437 339 308 49 262 988 494 406 50 191 536 287 359 51 381 770 455 439 52 321 591 360 422 53 195 789 389 397 54 325 499 326 301 55 413 702 441 472 56 179 786 381 328 57 180 357 212 249 58 206 662 343 359 59 255 414 264 318 60 380 698 426 437 61 226 465 273 317 62 370 462 329 388 63 265 874 450 388 64 163 279 175 245 65 302 1140 570 595 66 274 615 351 377 67 250 830 427 456 68 383 842 484 456 69 291 848 450 411 70 430 579 399 417 71 236 874 439 307 72 248 400 256 248 73 244 460 278 374 74 362 481 333 572 75 310 676 390 589 76 467 589 417 367 77 256 462 284 236 78 222 462 270 267 79 198 663 340 333 80 371 442 321 346 81 213 740 377 549 82 288 548 330 386 83 221 454 267 314 84 226 742 383 355 85 168 626 314 286 86 139 670 320 334 87 211 811 404 363 88 225 662 351 412 89 265 679 373 313 90 520 681 475 510 91 207 723 368 365 92 191 691 349 304 93 242 431 266 399 94 121 861 388 331 95 105 647 297 294 96 299 663 380 348 97 153 1040 471 441 98 242 336 228 260 99 288 396 270 448 100 530 798 525 494 101 178 494 266 211 102 410 758 462 343 103 491 646 387 401 104 443 603 413 408 105 218 742 379 314 106 322 496 323 347 107 404 897 514 420 108 217 659 346 281 109 277 563 332 294 110 200 438 252 229 111 258 495 298 276 112 523 829 534 440 113 283 486 304 408 114 260 444 278 242 115 231 750 388 389 116 481 609 431 405 117 293 969 499 370 118 261 375 282 311 119 152 335 192 227 120 291 302 234 282 121 383 629 400 440 122 295 640 370 295 123 276 440 283 304 124 113 1050 460 311 125 245 659 357 307 126 421 629 415 499 127 313 533 334 371 128 238 513 297 432 129 256 488 294 380 130 175 617 313 278 131 199 390 233 303 132 296 458 298 363 133 319 675 393 342 134 274 812 440 346 135 140 286 168 220 136 466 862 525 434 137 449 1530 782 739 138 252 489 293 282 139 330 421 297 440 140 165 313 189 215 141 292 609 356 389 142 306 688 393 473 143 345 403 296 292 144 359 880 466 492 145 399 506 358 449 146 593 1040 645 696 147 581 619 474 426 148 222 273 196 229 149 231 814 413 301 150 274 1360 646 460 151 178 1300 584 411 152 176 750 366 312 153 346 904 494 491 154 221 488 280 259 155 236 772 398 361 156 257 538 314 358 157 146 428 227 295 158 241 437 268 304 159 199 568 303 434 160 280 1530 715 506 161 370 476 334 417 162 148 628 307 250 163 246 700 374 284 164 263 602 342 349 165 178 674 337 275 166 393 712 437 432 167 193 437 249 246 168 148 397 215 214 169 120 596 290 217 170 262 573 330 307 171 369 647 402 366 172 394 431 326 415 173 289 949 489 396 174 282 461 294 329 175 225 785 399 336 176 255 1010 500 544 177 212 646 339 344 178 203 386 233 201 179 488 853 530 560 180 247 680 366 425 181 206 532 292 280 182 134 497 427 252 183 401 607 398 368 184 253 640 353 329 185 252 716 383 352 186 314 846 458 494 187 370 552 364 378 188 368 616 389 342 189 241 465 279 251 190 229 645 345 379 191 333 636 387 526 192 217 834 258 360 193 264 231 196 263 194 382 649 407 479 195 283 608 352 344 196 332 730 420 442 197 310 536 334 321 198 194 684 347 327 199 172 285 181 192 200 335 655 391 473

See FIG. 41.

Titration Model Results TBS-1-2010 Patient Simulation Data Testosterone Sample A taken 1 hour before the morning dose Testosterone Sample B taken 60 minutes after the morning dose Testosterone Model Concentration (ng/dL) Predicted Actual Subject Time From Morning Dose 24 h Cavg 24 h Cavg Number −1 hr +60 min (ng/dL) (ng/dL) 1 217 1400 225 501 2 286 896 238 393 3 406 372 315 358 4 449 822 324 392 5 367 970 262 514 6 254 554 178 334 7 406 846 312 584 8 483 684 395 450 9 108 212 103 175 10 260 739 212 366 11 327 373 287 322 12 522 1030 546 757 13 240 862 201 442 14 278 444 379 294 15 249 298 200 330 16 523 367 461 506 17 497 615 352 423 18 375 907 307 412 19 132 491 140 291 20 231 415 176 306 21 363 822 258 505 22 535 917 370 505 23 340 859 392 673 24 252 735 199 346 25 231 486 210 322 26 156 856 163 362 27 226 611 186 375 28 197 505 269 339 29 466 450 384 459 30 232 377 220 316 31 263 693 368 431 32 183 791 216 352 33 491 1020 339 436 34 277 762 199 475 35 362 371 320 441 36 140 543 117 233 37 164 502 174 301 38 195 303 184 209 39 381 1000 334 527 40 206 557 209 316 41 176 570 183 397 42 200 631 128 282 43 479 1040 385 877 44 280 466 260 332 45 212 229 220 315 46 246 482 188 300 47 117 286 116 203 48 420 386 255 308 49 262 830 220 406 50 191 601 233 359 51 381 405 337 439 52 321 420 263 422 53 195 677 129 397 54 325 364 282 301 55 413 697 315 472 56 179 1390 123 328 57 180 307 156 249 58 206 482 191 359 59 255 491 195 318 60 380 680 341 437 61 226 463 239 317 62 370 573 299 388 63 265 819 265 388 64 163 413 134 245 65 302 1110 246 595 66 274 409 236 377 67 250 609 191 456 68 383 643 314 456 69 291 548 227 411 70 430 966 298 417 71 236 497 185 307 72 248 486 169 248 73 244 665 183 374 74 362 605 318 572 75 310 571 313 589 76 467 488 308 367 77 256 462 190 236 78 222 277 176 267 79 198 459 168 333 80 371 424 229 346 81 213 1020 233 549 82 288 662 247 386 83 221 373 168 314 84 226 522 243 355 85 168 768 143 286 86 139 842 187 334 87 211 491 235 363 88 225 777 200 412 89 265 480 184 313 90 520 757 488 510 91 207 642 193 365 92 191 673 164 304 93 242 661 211 399 94 121 491 168 331 95 105 541 111 294 96 299 718 235 348 97 153 894 179 441 98 242 509 184 260 99 288 541 263 448 100 530 921 362 494 101 178 258 153 211 102 410 538 270 343 103 491 499 304 401 104 443 665 382 408 105 218 483 198 314 106 322 534 285 347 107 404 521 307 420 108 217 402 198 281 109 277 372 209 294 110 200 392 173 229 111 258 523 193 276 112 523 804 438 440 113 283 899 247 408 114 260 476 176 242 115 231 486 178 389 116 481 903 361 405 117 293 453 295 370 118 261 416 201 311 119 152 276 140 227 120 291 409 240 282 121 383 547 284 440 122 295 536 173 295 123 276 312 259 304 124 113 865 146 311 125 245 578 191 307 126 421 476 313 499 127 313 545 220 371 128 238 772 190 432 129 256 393 200 380 130 175 437 216 278 131 199 404 170 303 132 296 545 303 363 133 319 465 204 342 134 274 917 200 346 135 140 287 124 220 136 466 794 333 434 137 449 1030 292 739 138 252 347 176 282 139 330 445 321 440 140 165 271 144 215 141 292 460 268 389 142 306 637 217 473 143 345 419 253 292 144 359 852 327 492 145 399 690 292 449 146 593 1770 450 696 147 581 601 380 426 148 222 262 213 229 149 231 290 186 301 150 274 580 195 460 151 178 871 116 411 152 176 643 158 312 153 346 815 268 491 154 221 815 209 259 155 236 500 203 361 156 257 498 296 358 157 146 385 162 295 158 241 344 240 304 159 199 609 221 434 160 280 976 223 506 161 370 306 386 417 162 148 425 181 250 163 246 487 206 284 164 263 699 222 349 165 178 346 151 275 166 393 645 354 432 167 193 459 201 246 168 148 359 124 214 169 120 352 121 217 170 262 398 180 307 171 369 613 305 366 172 394 518 271 415 173 289 780 186 396 174 282 481 181 329 175 225 691 216 336 176 255 1190 278 544 177 212 466 229 344 178 203 209 150 201 179 488 367 375 560 180 247 501 208 425 181 206 483 152 280 182 134 455 99 252 183 401 605 283 368 184 253 352 199 329 185 252 470 181 352 186 314 721 287 494 187 370 789 281 378 188 368 277 255 342 189 241 522 182 251 190 229 711 175 379 191 333 645 326 526 192 217 387 181 360 193 264 472 198 263 194 382 401 421 479 195 283 726 191 344 196 332 582 305 442 197 310 534 252 321 198 194 726 200 327 199 172 286 184 192 200 335 322 363 473

See FIG. 42.

Titration Model Results TBS-1-2010 Patient Simulation Data Testosterone Sample A taken 1 hour before the morning dose Testosterone Sample B taken 90 minutes after the morning dose Testosterone Concentration Model (ng/dL) Predicted Actual Subject Time From Morning Dose 24 h Cavg 24 h Cavg Number −1 hr +90 min (ng/dL) (ng/dL) 1 217 860 726 501 2 286 633 531 393 3 406 407 349 358 4 449 276 571 392 5 367 949 600 514 6 254 846 363 334 7 406 1360 562 584 8 483 668 524 450 9 108 274 144 175 10 260 565 449 366 11 327 259 314 322 12 522 602 697 757 13 240 651 495 442 14 278 361 324 288 15 249 363 246 330 16 523 614 400 506 17 497 660 499 423 18 375 638 576 412 19 132 672 280 291 20 231 384 290 306 21 363 796 532 505 22 535 869 652 505 23 340 763 538 673 24 252 462 443 346 25 231 250 322 322 26 156 624 454 362 27 226 745 376 375 28 197 237 315 339 29 466 910 411 459 30 232 343 273 316 31 263 704 429 431 32 183 420 437 352 33 491 775 678 436 34 277 1610 466 475 35 362 714 329 441 36 140 212 307 233 37 164 322 299 301 38 195 325 224 209 39 381 1030 620 527 40 206 387 343 316 41 176 766 335 397 42 200 271 373 282 43 479 1500 682 877 44 280 537 335 332 45 212 361 198 315 46 246 342 327 300 47 117 246 181 203 48 420 416 362 308 49 262 812 490 406 50 191 415 356 359 51 381 880 353 439 52 321 755 333 422 53 195 917 392 397 54 325 375 309 301 55 413 472 498 472 56 179 907 704 328 57 180 269 219 249 58 206 490 309 359 59 255 413 335 318 60 380 555 476 437 61 226 494 309 317 62 370 385 423 388 63 265 627 409 388 64 163 228 259 245 65 302 709 634 595 66 274 550 307 377 67 250 477 386 456 68 383 635 461 456 69 291 666 377 411 70 430 636 627 417 71 236 561 329 307 72 248 345 330 248 73 244 554 408 374 74 362 443 434 572 75 310 762 396 589 76 467 317 429 367 77 256 368 322 236 78 222 417 224 267 79 198 585 295 333 80 371 402 357 346 81 213 917 554 549 82 288 426 427 386 83 221 702 267 314 84 226 747 336 355 85 168 455 420 286 86 139 622 440 334 87 211 401 315 363 88 225 658 450 412 89 265 378 334 313 90 520 653 573 510 91 207 525 381 365 92 191 472 388 304 93 242 694 405 399 94 121 605 275 331 95 105 437 290 294 96 299 359 457 348 97 153 504 470 441 98 242 363 337 260 99 288 281 372 448 100 530 593 651 494 101 178 378 196 211 102 410 286 426 343 103 491 530 444 401 104 443 634 497 408 105 218 454 315 314 106 322 390 384 347 107 404 680 415 420 108 217 309 278 281 109 277 319 291 294 110 200 538 266 229 111 258 342 351 276 112 523 774 596 440 113 283 715 531 408 114 260 612 330 242 115 231 468 322 389 116 481 394 621 405 117 293 431 335 370 118 261 358 304 311 119 152 355 192 227 120 291 448 314 282 121 383 673 418 440 122 295 310 373 295 123 276 368 264 304 124 113 494 439 311 125 245 444 370 307 126 421 524 403 499 127 313 626 385 371 128 238 952 453 432 129 256 499 291 380 130 175 344 275 278 131 199 472 271 303 132 296 301 378 363 133 319 522 352 342 134 274 464 535 346 135 140 626 192 220 136 466 531 566 434 137 449 1200 664 739 138 252 424 337 282 139 330 304 348 440 140 165 252 196 215 141 292 362 338 389 142 306 909 423 473 143 345 269 343 292 144 359 527 544 492 145 399 743 489 449 146 593 1080 1061 696 147 581 448 531 426 148 222 416 217 229 149 231 523 234 301 150 274 551 383 460 151 178 1150 471 411 152 176 775 368 312 153 346 667 521 491 154 221 265 340 259 155 236 771 330 361 156 257 558 339 358 157 146 309 238 295 158 241 341 263 304 159 199 528 363 434 160 280 916 564 506 161 370 487 304 417 162 148 380 257 250 163 246 413 329 284 164 263 510 432 349 165 178 421 235 275 166 393 770 466 432 167 193 475 293 246 168 148 262 228 214 169 120 368 212 217 170 262 616 296 307 171 369 606 441 366 172 394 598 409 415 173 289 475 480 396 174 282 427 343 329 175 225 524 411 336 176 255 864 649 544 177 212 531 304 344 178 203 253 185 201 179 488 516 384 560 180 247 546 336 425 181 206 396 309 280 182 134 468 264 252 183 401 663 452 368 184 253 282 272 329 185 252 584 324 352 186 314 750 465 494 187 370 425 520 378 188 368 392 290 342 189 241 234 343 251 190 229 390 422 379 191 333 695 439 526 192 217 695 271 360 193 264 321 330 263 194 382 564 352 479 195 283 630 453 344 196 332 531 410 442 197 310 323 379 321 198 194 454 413 327 199 172 290 206 192 200 335 533 295 473

See FIG. 43. Example 15 A Randomized 3-Way Cross Over Study to Assess the Relative Bioavailability, Safety and Tolerability of TBS-1 (4.5%) when Administered to Male Subjects with Seasonal Allergic Rhinitis in Symptomatic, Symptomatic but Treated (Oxymetazoline) and Asymptomatic States Using an Environmental Challenge Chamber (ECC) Model

-   -   Study Title: A randomized 3-way cross over study to assess the         relative bioavailability, safety and tolerability of TBS-1         (4.5%) when administered to male subjects with seasonal allergic         rhinitis in symptomatic, symptomatic but treated (oxymetazoline)         and asymptomatic states using an environmental challenge chamber         (ECC) model     -   Investigational Intranasal Testosterone Gel (TBS-1)     -   Products: Oxmetazoline Nasal Spray (0.05%) Dactylus gemerata         pollen (challenge substance)     -   EudraCT No.: 2011-006098-24     -   Development Phase: I (Extrinsic Factor Study)

Synopsis Study Title: Objectives: Primary:

The primary objective of this study was to determine and compare the pharmacokinetic (PK) profile of 11 mg TBS-1 (4.5%) administered intranasally 3 times a day in subjects who suffered from seasonal allergic rhinitis, whilst they were in the symptomatic, symptomatic but treated (with oxymetazoline) and asymptomatic states.

Secondary:

The secondary objective of this study was to determine and compare the local and systemic safety and tolerability, following 3 administrations of TBS-1 in subjects with seasonal allergic rhinitis, whilst they were in the above states.

Methodology:

This study was an open-label, balanced, randomized 3-way crossover, three-group, three-treatment, three-period pharmacokinetic study. Otherwise healthy male human subjects within the age range of 18 to 45 years with seasonal allergic rhinitis in an asymptomatic state were randomized to 1 of 3 sequence groups (A, B and C). Subjects in sequence group A received treatment 1 in period I, treatment 2 in period II and treatment 3 in period III. Subjects in sequence group B received treatment 2 in period I and treatment 3 in period II and treatment 1 in period III. Subjects in sequence group C received treatment 3 in period I, treatment 1 in period II and treatment 2 in period III. Subjects randomized to Treatment 1 (asymptomatic state) entered the ECC and were exposed to Dactylis glomerate pollen prior to each administration of TBS-1. Treatment 2 was administered to subjects who were in the symptomatic state of their diagnosed seasonal allergic rhinitis and were treated with oxymetazoline 30 min prior to the 07:00 h dose of TBS-1 and 12 hours ater the first administration. Subjects were exposed to Dactylis glomerate pollen in the ECC prior to each TBS-1 administration. Subjects receiving Treatment 3 were to be in the asymptomatic state (<3 for TNSS and <2 for the congestion score) and received three doses of TBS-1.

NUMBER OF SUBJECTS: Planned Sample Size: 18 Actual Sample Size: Safety Set: 18 Full PK population: 18 PK population for bioequivalence: 14

Name of Finished Product:

TBS-1

Name of Active Ingredient:

Testosterone 4.5% (TBS-1)

Diagnosis and Main Inclusion Criteria:

Diagnosis: otherwise healthy male human subjects with seasonal allergic rhinitis in asymptomatic state

Main Inclusion Criteria:

-   1. Otherwise healthy male human subjects within the age range of 18     to 45 years inclusive with seasonal allergic rhinitis in     asymptomatic state, which was defined by a positive case history and     a positive skin prick and/or intradermal test for Dactylis glomerata     pollen allergen within 12 months of screening. -   1. Total Nasal Symptom Score (TNSS) of ≥6/12 and a congestion score     of ≥2/3 on at least one card during the 2-hour screening challenge. -   2. Willingness to provide written informed consent to participate in     the study. -   3. Body-mass index of 30 kg/m². -   4. Absence of significant disease or clinically significant (cs)     abnormal laboratory values on laboratory evaluations, medical     history or physical examination during screening. -   5. Otorhinolaryngological examination without clinically significant     abnormal findings within 4 weeks of screening. -   6. Non-smokers or ex-smokers for at least six months.

Comprehension of the nature and purpose of the study and compliance with the requirement of the protocol.

STUDY DRUGS, DOSE AND MODE OF ADMINISTRATION, BATCH NUMBER: Study drug: Intranasal testosterone gel (TBS-1) Form and description: Multiple-dose dispenser Unit strength: 5.5 mg of 4.5% testosterone gel Daily dose: 33.0 mg of 4.5% testosterone gel Route of administration: intranasal Posology: t.i.d. Batch number: 2372 Oxymetazoline (Nasivin ® ohne Study drug: Konservierungsstoffe) Form and description: Multiple-dose dispenser Unit strength: 0.05% oxymetazoline hydrochloride Daily dose: 4 puffs (2 per nostril) of 5.05% oxymetazoline hydrochloride Route of administration: Intranasal Posology: 30 min prior to the 07:00 h dose of TBS-1 and 12 h after the first dose (during Treatment Sequence 3) Study drug: Dactylis glomerata (pollen) Form and description: Challenge substance for pollen chamber Unit strength: 6 g Daily dose: 4000 ± 500 of Dactylis glomerate pollen Route of administration: Inhalation STUDY PERIOD: 5 weeks

Criteria for Evaluation: Primary Endpoint (Pharmakokinetics):

The following pharmacokinetic (PK) parameters were determined for all subjects in all treatments: Area under the serum concentration time plot up to 24 h (AUC₀₋₂₄), the average of the observed concentration of testosterone and DHT in the 24 h interval (C_(avg)), minimum observed concentration of testosterone and DHT (C_(min)), maximum observed concentration of testosterone and DHT (C_(max)), and time of maximum observed concentration testosterone and DHT (t_(max)) for 3 treatment phases (Treatments 1-3). The relative PK profiles of the 3 treatments were determined using AUC₀₋₂₄ and C_(max) corrected for the serum testosterone concentration.

Secondary Endpoint (Safety):

Safety and tolerability were assessed by monitoring:

-   -   Adverse events     -   Otolaryngological examination     -   Vital signs     -   Complete blood count to evaluate changes in white blood cell         (WBC) count, hemoglobin and hematocrit     -   Clinical chemistry profile     -   Urinalysis (urine specific gravity, glucose, protein, ketone,         pH, blood, bilirubin, urobilinogen, nitrite, leukocytes)

Statistical Methods:

Continuous measurements were summarized by means of descriptive statistics (i.e., number of observations, arithmetic mean, standard deviation [SD], minimum, median, maximum). Categorical variables were summarized by means of frequency tables (i.e. count and percentages). All baseline corrected PK parameters were tested regarding bioequivalence (ANOVA).

Summary—Conclusions

The 18 treated subjects were aged between 27 and 44. All 6 subjects in sequence group A completed the study as scheduled. In sequence group B, 4 out of 6 subjects and sequence group C, 5 out of 6 subjects completed the study as scheduled.

Pharmacokinetic Conclusion:

Administration of TBS-1 under asymptomatic, symptomatic and symptomatic but treated conditions of allergic rhinitis demonstrated a reliable increase in testosterone serum concentrations in all three treatment groups. The drug induced exposure to testosterone and DHT, determined as AUC_(0-24,bc) was higher in the asymptomatic state compared to symptomatic and symptomatic but treated state. ANOVA analysis failed to demonstrate bioequivalence between the asymptomatic state and either symptomatic or symptomatic but treated state.

A comparison of the AUC_(bc) over 0-24 h between symptomatic and symptomatic but treated state revealed no bioequivalence between these two treatment conditions.

However, given that the point estimates were close to 1 (1.0903 for testosterone and 0.9944 for DHT) the failure to show bioequivalence may be due to large inter-individual variations. These large variations led to wide confidence intervals, which exceed the threshold values for bioequivalence of 0.8 to 1.25.

While TBS-1 bioavailability during the symptomatic state of allergic rhinitis is lower than during the asymptomatic state, the post-dose concentrations of testosterone still demonstrate a reliable increase in levels as compared to baseline. The relative decrease in bioavailability of TBS-1 under symptomatic seasonal rhinitis is not either ameliorated or aggravated by the administration of oxymetazoline.

Safety Conclusion:

TBS-1 was well tolerated. All reported AEs were of mild or moderate intensity and all were transient. All reported AEs were deemed treatment emergent with no causality to TBS-1. Physical examination, vital signs and clinical laboratory results did not reveal any clinically significant finding.

Example 16 An Open Label, Randomized, Balanced, Three Treatments, Parallel Design, Pharmacokinetic Study of Intra-Nasal TBS-1 Administration to Hypogonadal Men Pharmacokinetic Simulation Report

See Exhibit D (the contents of which are incorporated herein by reference).

It should be understood that the present invention contemplates any effective pharmacokinetic parameter for the intra-nasal TBS-1 gels of the present invention, including those that may vary as much as about ±25% of the pharmacokinetic parameters set forth in Exhibit D. Preferably, the present invention contemplates pharmacokinetic parameters for the intra-nasal TBS-1 gels that are about 25% greater and/or about 20% lesser than those pharmacokinetic parameters set forth in Exhibit D.

Example 17 Stability Intra Nasal Testosterone Gels and Diffusion Rates

The present invention also contemplates stable intranasal TBS-1 testosterone gels as set forth in Exhibits F, G, H, I, J, K1, K2, L, M1, M2 and M3 (the contents of which are incorporated herein by reference) and intranasal TBS-1 testosterone gels having diffusion rates as set forth in Exhibit N (the contents of which are incorporated herein by reference).

Example 18 A Randomized 3-Way Cross Over Study to Assess the Relative Bioavailability, Safety and Tolerability of 4.5% TBS-1 when Administered to Male Subjects with Seasonal Allergic Rhinitis

Amount (% w/w) Component 4.5% TBS-1 Testosterone  4.5% Castor Oil, USP 87.5% Oleoyl polyoxylglycerides, Ph Eur/NF  4.0% Colloidal silicon dioxide, NF  4.0%

Composition of TBS-1

This study assessed the relative bioavailability, safety and tolerability of 4.5% TBS-1 when administered to patients with symptomatic untreated and treated (oxymetazoline) seasonal allergic rhinitis as well as asymptomatic subjects using an environmental challenge chamber (ECC) model.

The purpose of this study was to determine effect of allergic rhinitis and the treatment of allergic rhinitis, oxymetazoline, on the absorption of TBS-1. This was achieved by determining the testosterone pharmacokinetic profile following administration of 11 mg TBS-1 (4.5%) three times a day in subjects that suffer from seasonal allergic rhinitis, while in the symptomatic, symptomatic but treated (with Oxymetazoline) and asymptomatic states. The secondary objective of the study was to determine the local and systemic safety and tolerability, following three administrations of TBS-1 in subjects with seasonal allergic rhinitis and while taking oxymetazoline.

Symptoms of allergic rhinitis were induced in 18 male patients using allergen challenge with Dactylis glomerate pollen in and Environmental Challenge Chamber. The study was a 3-period cross over design in which all subjects received each of the following treatments:

A: TBS-1 (Symptomatic State)

Symptoms of allergic rhinitis were induced in men with seasonal allergic rhinitis by exposing them to pollen of Dactylis glomerate in an environmental challenge chamber (ECC) prior to each administration of TBS-1.

B: TBS-1 and Oxymetazoline (Symptomatic and Treated)

Oxymetazoline nasal spray was administered 30 minutes prior to the 0700 hr dose of TBS-1 and again 12 hrs after the first dose. Symptoms of allergic rhinitis were induced in men with seasonal allergic rhinitis by exposing them to pollen in an Environmental Challenge Chamber.

C: TBS-1 (Asymptomatic State)

TBS-1 was administered 3 times a day to men in the asymptomatic state.

This is a single site study with a planned enrolment of 18 healthy men. A 24 hour pharmacokinetic profile of testosterone and DHT will be performed on all subjects in all treatments.

Safety Results

Eighteen (18) healthy men with allergic rhinitis were exposed to TBS-1. TBS-1 was well tolerated by subjects. There were no deaths in the study and none of the subjects experienced any SAEs. Fifteen (15) adverse events were encountered in the study: 2 in asymptomatic state; 6 in the symptomatic state; and 7 in the symptomatic but treated state. None of the adverse events were considered related to the study drug. All events were of mild to moderate severity. None of the subjects were discontinued from the treatment because of an AE (see results in the following table).

TABLE Adverse Events Unrelated to TBS-1 Symptomatic Asymptomatic Symptomatic but treated State state state Event (n = 18) (n = 15) (n = 17) Musculoskeletal and connective tissue disorder Musculosketal 1 (5.6%) stiffness Respiratory, thoracic and mediastinal disorder Epistaxis 1 (6.7%) Dysphonia 1 (6.7%) 2 (11.8%) Oropharyngeal pain 1 (6.7%) Rhinitis allergic 1 (6.7%) Invesigations Forced expiry volume 1 (6.7%) 1 (5.9%) decreased General disorders and administration site condition Injection site phlebitis 2 (13.3%) 1 (5.9%) Infections and infestations Nasopharyngitis 2 (11.8%) Nervous system disorders Dizziness 1 (5.9%)

Test results are also presented in Exhibit M (the contents of which are incorporated herein by reference).

Example 19 A Randomized 3 Way Cross Over Study to Assess Relative Bioavailability, Safety and Tolerability of 4.5% TBS 1TBS-1 (4.5%) when Administered to Male Subjects with Seasonal Allergic Rhinitis in Symptomatic, Symptomatic but Treated (Oxymetazoline) and Asymptomatic States

An environmental challenge chamber (ECC) model was used in this study.

Objectives:

The primary objective of this study was to determine and compare the pharmacokinetic (PK) profile of 11 mg TBS-1 (4.5%) administered intranasally 3 times a day in subjects who suffered from seasonal allergic rhinitis, whilst they were in the symptomatic, symptomatic but treated (with oxymetazoline) and asymptomatic states.

The secondary objective of this study was to determine and compare the local and systemic safety and tolerability, following 3 administrations of TBS-1 in subjects with seasonal allergic rhinitis, whilst they were in the above states.

General Study Design:

The chosen cross over design allows to control for non-treatment effects such as period and sequence. Intra-individual measurements allow to detect treatment effects with a higher sensitivity as compared to inter-individual measurements based on smaller intra-individual variation.

This was an open-label study, as the physical differences in the intranasal dosing devices prevent blinding. Since pharmacokinetic parameters are objective measures, they were likely not affected by the open-label design of the study.

Methodology:

This study was an open-label, balanced, randomized 3-way crossover, three-group, three-treatment, three-period pharmacokinetic study. Otherwise healthy male human subjects within the age range of 18 to 45 years with seasonal allergic rhinitis in an asymptomatic state were randomized to 1 of 3 sequence groups (A, B and C).

Subjects in sequence group A received treatment 1 in period I, treatment 2 in period II and treatment 3 in period III. Subjects in sequence group B received treatment 2 in period I and treatment 3 in period II and treatment 1 in period III. Subjects in sequence group C received treatment 3 in period I, treatment 1 in period II and treatment 2 in period III (as shown in the following table).

Treatments in the Three Dose Sequences PERIOD I PERIOD II PERIOD III Visit 3 Visit 4 Visit 5 Time 04:00-07:00 04:00-07:00 04:00-07:00 (+1 day) (+1 day) (+1 Day) Sequence group A Treatment 1 Treatment 2 Treatment 3 Sequence group B Treatment 2 Treatment 3 Treatment 1 Sequence group C Treatment 3 Treatment 1 Treatment 2

Subjects randomized to Treatment 1 (asymptomatic state) entered the ECC and were exposed to Dactylis glomerata pollen prior to each administration of TBS-1. Treatment 2 was administered to subjects who were in the symptomatic state of their diagnosed seasonal allergic rhinitis and were treated with oxymetazoline 30 min prior to the 07:00 h dose of TBS-1 and 12 hours after the first administration. Subjects were exposed to Dactylis glomerata pollen in the ECC prior to each TBS-1 administration. Subjects receiving Treatment 3 were to be in the asymptomatic state (<3 for TNSS and <2 for the congestion score) and received three doses of TBS-1.

Number of Subjects: 18

Safety Set:

Full PK population: 18

PK population for Bioequivalence: 14

Subject Population:

A male subject population with a history of seasonal allergic rhinitis, aged 18-45 years was chosen for this study in order to investigate the effect of allergic rhinitis on the absorption of TBS-1 in an asymptomatic, symptomatic and symptomatic but treated state.

Diagnosis criteria: otherwise healthy male human subjects with seasonal allergic rhinitis in asymptomatic state.

Main Inclusion Criteria:

1. Otherwise healthy male human subjects within the age range of 18 to 45 years inclusive with seasonal allergic rhinitis in asymptomatic state, which was defined by a positive case history and a positive skin prick and/or intradermal test for Dactylis glomerate pollen allergen within 12 months of screening.

2. Total Nasal Symptom Score (TNSS) of ≥6/12 and a congestion score of ≥2/3 on at least one card during the 2-hour screening challenge.

3. Willingness to provide written informed consent to participate in the study.

4. Body-mass index of 30 kg/m².

5. Absence of significant disease or clinically significant (cs) abnormal laboratory values on laboratory evaluations, medical history or physical examination during screening.

6. Otorhinolaryngological examination without clinically significant abnormal findings within 4 weeks of screening.

7. Non-smokers or ex-smokers for at least six months.

8. Comprehension of the nature and purpose of the study and compliance with the requirement of the protocol.

-   Study drug: Intranasal testosterone gel (TBS-1) -   Form and description: Multiple-dose dispenser -   Unit strength: 5.5 mg of 4.5% testosterone gel -   Daily dose: 33.0 mg of 4.5% testosterone gel -   Route of administration: intranasal -   Posology: t.i.d. -   Batch number: 2372 -   Study drug: Oxymetazoline (Nasivin® ohne Konservierungsstoffe) -   Form and description: Multiple-dose dispenser -   Unit strength: 0.05% oxymetazoline hydrochloride -   Daily dose: 4 puffs (2 per nostril) of 5.05% oxymetazoline     hydrochloride -   Route of administration: Intranasal -   Posology: 30 min prior to the 07:00 h dose of TBS-1 and 12 h after     the first dose (during Treatment Sequence 3) -   Study drug: Dactylis glomerata (pollen) -   Form and description: Challenge substance for pollen chamber -   Unit strength: 6 g -   Daily dose: 4000±500 of Dactylis glomerata pollen -   Route of administration: Inhalation -   Study Period: 5 Weeks

Criteria for Evaluation:

Primary Endpoint (Pharmakokinetics):

The following pharmacokinetic (PK) parameters were determined for all subjects in all treatments: Area under the serum concentration time plot up to 24 h (AUC₀₋₂₄), the average of the observed concentration of testosterone and DHT in the 24 h interval (C_(avg)), minimum observed concentration of testosterone and DHT (C_(min)), maximum observed concentration of testosterone and DHT (C_(max)), and time of maximum observed concentration testosterone and DHT (t_(max)) for 3 treatment phases (Treatments 1-3).

The relative PK profiles of the 3 treatments were determined using AUC₀₋₂₄ and C_(max) corrected for the serum testosterone concentration.

Secondary Endpoint (Safety):

Safety and tolerability were assessed by monitoring:

-   -   Adverse events     -   Otolaryngological examination     -   Vital signs     -   Complete blood count to evaluate changes in white blood cell         (WBC) count, hemoglobin and hematocrit     -   Clinical chemistry profile     -   Urinalysis (urine specific gravity, glucose, protein, ketone,         pH, blood, bilirubin, urobilinogen, nitrite, leukocytes)

Statistical Methods:

Continuous measurements were summarized by means of descriptive statistics (i.e., number of observations, arithmetic mean, standard deviation [SD], minimum, median, maximum). Categorical variables were summarized by means of frequency tables (i.e. count and percentages). All baseline corrected PK parameters were tested regarding bioequivalence (ANOVA).

Subjects participating in this study were at risk for the side effects common to all formulations of testosterone. In addition to risks inherent to all testosterone administration, subjects receiving TBS-1 in prior clinical studies have experienced mild nasal symptoms including dryness, inflammation, congestion, and discomfort. None of these AEs prevented subjects from continuing the medication.

The exposure to pollen in order to induce symptoms of allergic rhinitis was associated with a minimal risk of anaphylactic reactions. Allergen challenges with Dactylis glomerate pollen in the Fraunhofer ECC were designed to mimic the situation for the subject under quasi-natural conditions. Therefore, the pollen exposure in the ECC did not present a greater risk than natural exposure during the grass pollen season in summer. The experimental setting was validated and used in numerous clinical trials. Inhalation of pollen can cause bronchoconstriction in asthmatic subjects. However, asthmatic subjects were excluded from the study. For risk minimization measures with respect to pollen challenge.

Subjects receiving oxymetazoline (Nasivin©) were at risk of the described side-effects of this product. Frequent side-effects are burning and dryness of the nasal mucosa and sneezing. Uncommon side effects are agitation, fatigue, headache, hallucinations (mainly observed in children), tachycardia, hypertension, arrhythmia, nose bleeding, convulsions (mainly observed in children) and hypersensitivity reactions, such as, itching and rash. However, since each subject received only 2 doses of oxymetazoline, the risk of developing side-effects was minimal.

Testosterone replacement therapy for hypogonadal men should correct the clinical abnormalities of testosterone deficiency. Since this was a Phase I study enrolling men not suffering from hypogonadism between the ages of 18-45 years it was not anticipated that these volunteers would directly benefit by taking part in this study.

Conclusions:

The 18 treated subjects were aged between 27 and 44. All 6 subjects in sequence group A completed the study as scheduled. In sequence group B, 4 out of 6 subjects and sequence group C, 5 out of 6 subjects completed the study as scheduled.

Pharmacokinetic Conclusion:

Administration of TBS-1 under asymptomatic, symptomatic and symptomatic but treated conditions of allergic rhinitis demonstrated a reliable increase in testosterone serum concentrations in all three treatment groups. The drug induced exposure to testosterone and DHT, determined as AUC_(0-24,bc) was higher in the asymptomatic state compared to symptomatic and symptomatic but treated state. ANOVA analysis failed to demonstrate bioequivalence between the asymptomatic state and either symptomatic or symptomatic but treated state.

A comparison of the AUC_(bc) over 0-24 h between symptomatic and symptomatic but treated state revealed no bioequivalence between these two treatment conditions. However, given that the point estimates were close to 1 (1.0903 for testosterone and 0.9944 for DHT) the failure to show bioequivalence may be due to large inter-individual variations. These large variations led to wide confidence intervals, which exceed the threshold values for bioequivalence of 0.8 to 1.25.

Administration of 4.5% TBS-1 under asymptomatic, symptomatic and symptomatic but treated conditions of allergic rhinitis demonstrated a reliable increase in testosterone serum concentrations under all three treatment conditions. 4.5% TBS 1 bioavailability during the symptomatic state of allergic rhinitis was 21% lower compared to the asymptomatic state, based on AUC0-24 values.

While TBS-1 bioavailability during the symptomatic state of allergic rhinitis is lower than during the asymptomatic state, the post-dose concentrations of testosterone still demonstrate a reliable increase in levels as compared to baseline. The relative decrease in bioavailability of 4.5% TBS 1 under symptomatic seasonal rhinitis was neither ameliorated nor aggravated by the administration of oxymetazoline.

Safety Conclusion:

TBS-1 was well tolerated. All reported AEs were of mild or moderate intensity and all were transient. All reported AEs were deemed treatment emergent with no causality to TBS-1. Physical examination, vital signs and clinical laboratory results did not reveal any clinically significant finding.

See FIGS. 44 and 45 respectively

Example 20 Drug-Drug Interaction Study to Evaluate Administration Route of Intranasal Application of Testosterone and to Investigate Potential Interaction of Testosterone with a Nasal Decongestant Spray

A drug-drug Interaction study was completed, which was an extrinsic factor study to evaluate whether intranasal application of testosterone is a reliable route of administration during naturally occurring nasal inflammation such as allergic rhinitis and to investigate the potential interaction of TBS-1 with a nasal decongestant spray, oxymetazoline. The study was conducted at one site in Germany.

Treatment Regimen.

Subjects were randomly assigned to a treatment sequence comprised of TBS-1 when they were asymptomatic, symptomatic and untreated and symptomatic and treated with oxymetazoline nasal spray. The symptomatic state was induced by exposure to Dactylis glomerata pollen in an environment exposure chamber (EEC).

The symptomatic state was defined by a positive case history, a positive skin prick and/or interdermal test for Dactylis glomerata allergen and a Total nasal Symptom Score (TNSS) of ≥6/12 and a congestion score of ≥2/3. TBS-1 administration to subjects in a symptomatic and treated arm received oxymetazoline 30 minutes prior to the 07:00 hour dose of TBS 1 and 12 hours after the first administration. All patients received 3 doses of TBS-1 at 07:00, 13:00 and 21:00 hrs.

Primary Objective

The primary objective of this study was to determine and compare the pharmacokinetic (PK) profile of 11 mg TBS-1 (4.5%) administered intranasally 3 times a day in subjects who suffered from seasonal allergic rhinitis, whilst they were in the symptomatic, symptomatic but treated (with oxymetazoline) and asymptomatic states.

Subject Disposition

The 18 treated subjects were healthy subjects with seasonal allergic rhinitis aged between 27 and 44. All 6 subjects in sequence group A completed the study as scheduled. In sequence group B, 4 out of 6 subjects and sequence group C, 5 out of 6 subjects completed the study as scheduled. In total, the number of subjects completing each of the 3 states were: asymptomatic (N=18), symptomatic but treated (N=17), and symptomatic untreated (N=15).

Analysis of Primary Endpoint

Administration of TBS-1 under asymptomatic, symptomatic and symptomatic but treated conditions of allergic rhinitis demonstrated a reliable increase in testosterone serum concentrations under all 3 treatment conditions as presented in the following table and the following figure.

TABLE AUC Values for Serum Testosterone by Treatment Condition Including Non-Corrected Values, Corrected Values and Pre-dose Corrected Values Pre-dose Non-corrected Corrected Corrected Values Values* Values^(#) Asymptomatic 16746 ± 3894 5797 ± 2643 3841 ± 2713 Symptomatic 13217 ± 3589 2267 ± 2172 3041 ± 1967 Symptomatic but 12778 ± 3379 1828 ± 1889 3138 ± 1480 treated *Corrected values = uncorrected values-baseline 24 hour ^(#)Pre-dose corrected values = PK values were corrected for treatment specific pre-dose levels

See FIG. 46.

FI Serum Testosterone (ng/dL): Arithmetic Mean Concentration vs. Time Curve, Linear Scale (PK set)

The testosterone exposure as estimated by the mean baseline-corrected area under the serum concentration-time curve from 0 to 24 hours post-dose AUC_(0-24,bc) was higher for subjects in the asymptomatic state compared to symptomatic and symptomatic but treated state. An analysis of variance did not demonstrate bioequivalence between the asymptomatic state and either symptomatic and symptomatic but treated state.

The difference in AUC_(0-24,bc) between the symptomatic untreated and the symptomatic treated states was small, indicating that administration of oxymetazoline did not relevantly affect the absorption of TBS-1; however, they were not bioequivalent. Given that the point estimates were close to 1 (1.0903) the failure to show bioequivalence may be due to large interindividual variations. These large variations led to wide confidence intervals, which exceed the threshold for bioequivalence of 0.8 to 1.25.

TBS 1 bioavailability during the symptomatic state of allergic rhinitis was 21% lower compared the asymptomatic state, based on AUC₀₋₂₄ values. However, the post-dose concentrations of testosterone still demonstrate a reliable increase in levels as compared to baseline. The relative decrease in bioavailability of TBS-1 under symptomatic seasonal rhinitis is neither ameliorated nor aggravated by the administration of oxymetazoline.

Additional exploratory analysis revealed that the different treatment conditions influenced the pre-dose value of testosterone. A student t-test showed significant differences in the pre-dose testosterone between the asymptomatic treatment condition compared to the symptomatic and the symptomatic and treated conditions. Subjects were exposed to an EEC in the symptomatic and symptomatic and treated condition but not in the asymptomatic condition. It is hypothesized that the earlier wake up time and/or stress caused by procedures associated with confinement in the EEC may have led to lower testosterone values in both symptomatic states compared to the asymptomatic state. As such, the baseline profile collected under the EEC conditions and used for correction purposes was not truly representative of the non-treated state under all study conditions. The additional analysis corrected for endogenous testosterone by pre-dose values instead of correction by 24 hour baseline profile. This analysis showed that the differences between asymptomatic and both symptomatic treatment conditions were less pronounced with respect to AUC_(bc), C_(avg,bc), and C_(max,bc). However, bioequivalence could not be shown between treatment conditions.

REFERENCE LIST

-   1. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics,     4th edition, 2006. Editors; Burtis C A, Ashwood E R, and Bruns D E. -   2. Wang C, Swerdloff RS. Androgen replacement therapy. Ann Med 1997;     29: 365-370. -   3. Matsumoto A M. Andropause: clinical implications of the decline     in serum Testosterone levels with aging in men. J Gerontol A Med Sci     2002; 57: M76-M99. -   4. Haren M T, Kim M J, Tariq S H, Wittert G A, Morley J E.     Andropause: a quality-of-life issue in older males. Med Clin North     Am 2006; 90: 1005-1023. -   5. Nieschlag E. Testosterone treatment comes of age: new options for     hypogonadal men. Clin Endocrinol (Oxf) 2006: 65: 275-281. -   6. Tenover J L. The androgen-deficient aging male: current treatment     options. Rev Urol 2003; 5 (Suppl): S22-S28. -   7. Jockenhovel F. Testosterone therapy—what, when and to whom? Aging     Male 2004; 7: 319-324. -   8. Kunz G H, Klein K O, Clemons R D, Gottschalk M E, Jones K L.     Virilization of young children after topical androgen use by their     parents. Pediatrics 2004; 114: 282-284. -   9. Brachet C, Vermeulen J, Heinrichs C. Children's virilisation and     the use of a Testosterone gel by their fathers. Eur J Pediatr 2005;     164: 646-647. -   10. Bagchus W M, Hust R, Maris F, Schnabel P G, Houwing N S.     Important effect of food on the bioavailability of oral Testosterone     undecanoate. Pharmacotherapy 2003; 23: 319-325. -   11. Haren M, Chapman I M, Haren M T, MacKintosh S, Coates P, Morley     J E. Oral Testosterone supplementation increases muscle and     decreases fat mass in healthy elderly males with low normal gonadal     status. J Gerontol A Biol Sci Med Sci 2003; 58: 618-625. -   12. Haren M, Chapman I, Coates P, Morley J E, Wittert G. Effect of     12 month oral Testosterone on Testosterone deficiency symptoms in     symptomatic elderly males with low-normal gonadal status. Age Ageing     2005; 34: 123-130. -   13. Mattern C, Hoffmann C, Morley J E, Badiu C. The Aging Male 2008;     11: 171-178. 

1. A method of treating a male, who is experiencing symptomatic allergic or seasonal rhinitis, with testosterone replacement therapy for a condition associated with a deficiency or absence of endogenous who is in need of such testosterone replacement treatment, the method comprising: administering intranasally to the symptomatic male three times a day an intranasal testosterone gel to deliver intranasally to the symptomatic male a total daily dose of about 33 mg of testosterone to effectively treat said condition in the symptomatic male wherein said condition is hypogonadism.
 2. The intranasal testosterone method claim 1, wherein the intranasal method includes the further step of: intranasally administering to the symptomatic male nasal vasoconstrictor and/or a nasal decongestant to intranasally treat the symptomatic male with a combination of the testosterone and the nasal vasoconstrictor and/or nasal decongestant.
 3. The intranasal testosterone method of claim 2, wherein said nasal vasoconstrictor or said nasal decongestant comprises a therapeutic agent selected from the group of ephedrine, levomethamphetamine, aphazoline, oxymetazoline, phenylephrine, pseudoephedrine, tramazoline, and xylometazoline.
 4. The intranasal testosterone method of claim 2, wherein the nasal vasoconstrictor or the nasal decongestant comprises oxymetazoline.
 5. The intranasal testosterone method of claim 4, wherein the intranasal oxymetazoline method step comprises administering the oxymetazoline intranasally prior to said intranasal testosterone administration.
 6. The intranasal testosterone method of claim 1, wherein, when the oxymetazoline is intranasally administered about 30 minutes prior to said intranasal testosterone administration, a decrease of about 2.6% in mean testosterone AUC and a decrease of about 3.6% in mean C_(max) of total testosterone is observed in the symptomatic male as compared to when the symptomatic male is left untreated with the oxymetazoline.
 7. The intranasal testosterone method of claim 1, wherein said intranasal testosterone gel comprises: a. about 4.5% testosterone by weight of said intranasal gel; and b. a pharmaceutically acceptable vehicle.
 8. (canceled)
 9. The intranasal testosterone method of claim 1, wherein said intranasal testosterone gel comprises a solvent, a wetting agent, and a viscosity increasing agent.
 10. The intranasal testosterone method of claim 9, wherein the solvent is castor oil.
 11. The intranasal testosterone method of claim 9, wherein said wetting agent is an oleoyl polyoxylglyceride.
 12. The intranasal testosterone method of claim 9, wherein said viscosity increasing agent is colloidal silicon dioxide.
 13. The intranasal testosterone method of claim 1, wherein said intranasal testosterone gel comprises castor oil, oleoyl polyoxylglycerides and colloidal silicon dioxide.
 14. The intranasal testosterone method of claim 1, wherein the symptomatic male has a testosterone C_(avg) within the normal testosterone range of 300 to 1050 ng/dL on or after day 90 of said intranasal testosterone treatment.
 15. The intranasal testosterone method of claim 1, wherein the symptomatic male does not have a testosterone C_(max) of greater than 2500 ng/dL on or after day 90 of said intranasal testosterone treatment.
 16. The intranasal testosterone method of claim 1, wherein said intranasal testosterone gel has a testosterone rate of diffusion of between about 28 to about 100 slope/mgT %.
 17. The intranasal testosterone method of claim 16, wherein said testosterone diffusion rate is between about 30 to about 95 slope/mgT %.
 18. The intranasal testosterone method of claim 16, wherein said testosterone diffusion rate is between about 28 to about 35 slope/mgT %.
 19. The intranasal testosterone method of claim 1, wherein said intranasal testosterone gel comprises 4.5% testosterone, 87.5% castor oil, 4.0% oleoyl polyoxylglycerides, and 4.0% colloidal silicon dioxide.
 20. The intranasal testosterone method of claim 1, wherein said intranasal testosterone method includes the further step of: providing a metered dose pump containing 11 grams of the intranasal testosterone gel, wherein the metered dose pump dispenses 60 metered pump actuations, wherein one pump actuation delivers 5.5 mg of the testosterone in 0.122 grams of the intranasal testosterone gel, so that, when the symptomatic male actuates the one said pump from the metered dose pump into each nostril of the symptomatic male three times a day, the total daily dose of about 33 mg of testosterone is intranasally administered to the symptomatic male.
 21. An intranasal testosterone method for treating a male, who is symptomatic for allergic or seasonal rhinitis, with testosterone replacement therapy for a condition associated with a deficiency or absence of endogenous testosterone that is in need of such testosterone replacement treatment, said method comprising: providing a metered dose pump containing 11 grams of an intranasal testosterone gel, wherein the metered dose pump dispenses 60 metered pump actuations of the intranasal testosterone gel; and instructing the symptomatic male to actuate one pump from the metered dose pump into each nostril of the symptomatic male three times a day to deliver intranasally to the symptomatic male a total daily dose of about 33 mg of testosterone to effectively treat the symptomatic male for the condition; wherein the one pump actuation delivers 5.5 mg of the testosterone in 0.122 grams of the intranasal testosterone gel into each of the nostrils; wherein the condition is hypogonadism; and wherein the hypogonadism is selected from a group of hypogonadisms consisting of congenital or acquired primary hypogonadism and congenital or acquired hypogonadotropic hypogonadism. 